Compositions comprising MIR34 therapeutic agents for treating cancer

ABSTRACT

In one aspect, the invention generally relates to compositions comprising miR-34 and siRNAs functionally and structurally related to miR-34 for the treatment of cancer.

FIELD OF THE INVENTION

The invention generally relates to compositions comprising miR-34 andsiRNAs functionally and structurally related to miR-34 for the treatmentof cancer.

BACKGROUND

The following is a discussion of relevant art pertaining to TP53 andRNAi. The discussion is provided only for understanding of the variousembodiments of invention that follow. The summary and references citedthroughout the specification herein are not an admission that any of thecontent below is prior art to the claimed invention.

The TP53 tumor suppressor is activated by protein stabilizationfollowing genotoxic stress. This activation can be induced byultraviolet or ionizing radiation as well as a host of DNA-damagingchemotherapeutics such as doxorubicin (adriamycin), cisplatin, andbleomycin. Activation of TP53 leads to cell cycle arrest prior to entryinto S phase and/or apoptosis. TP53 activation also initiates a numberof DNA repair pathways (Fei and El'Deiry, 2003, Oncogene 22:5774-83).Mutations in TP53, which are present in about 50% of human cancers(Hollstein et al., 1991, Science 253:49-53), result in checkpointdefects and may contribute to uncontrolled cell proliferation, genomicinstability, and accumulation of tumorigenic mutations (Prives and Hall,1999, J. Pathol. 186:112-26). In the clinic, emphasis has been placed onidentifying chemotherapeutics that are effective for both TP53-positivetumor cells and TP53-deficient tumor cells (Lowe et al., 1994, Science266:807-810; Lacroix et al., 2006, Endocrine-Related Cancer 13:293-325;Levesque and Eastman, 2007, Carcinogenesis 28:13-20). Therefore,predicting TP53 pathway status in human tumors will be an importantcomponent for selecting an effective cancer therapeutic for a givencancer type.

Although DNA sequencing of TP53 can reveal inactivating mutations, theTP53 pathway can be inactivated by alternative mechanisms. For example,p19(ARF), which is encoded by the INK4a-ARF locus, inhibits cellproliferation by activating TP53 (Sherr et al., 2005, Cold Spring HarborSymp. Quant. Biol. 70:129-37). Significantly, many human cancers exhibitdeletion, silencing, or mutation of the INK4a-ARF locus. Other tumorsover-express, or express aberrant splice forms of, MDM2, a key regulatorof TP53 stability and transcriptional activity (Levav-Cohen et al.,2005, Growth Factors 23:183-92). TP53 pathway inactivation can also becaused by viral factors such as the human papilloma virus E6 protein,which binds to and targets TP53 for degradation. Therefore, predictingTP53 pathway integrity may not be straightforward in many patienttumors. Miller et al. (2005, PNAS 38:13550-55) developed a geneexpression signature to predict TP53 pathway status of cancer patientsand presented data showing the importance of TP53 pathway status inpredicting clinical breast cancer behavior.

There is growing realization that miRNAs, in addition to functioning asregulators of development, can act as oncogenes and tumor suppressors(Akao et al., 2006, Oncology Reports 16:845-50; Esquela-Kerscher andSlack, 2006, Nature Rev., 6:259-269; He et al., 2005, Nature 435:828-33)and that miRNA expression profiles can, under some circumstances, beused to diagnose and classify human cancers (Lu et al., 2005, Nature435:834-38; Volinia et al., 2006, PNAS 103:2257-61; Yanaihara et al.,2006, Cancer Cell 9:189-198). Given the significance of TP53 in cancerand the importance of finding clinical biomarkers for TP53 status, thereis need to identify RNA transcripts, including miRNAs, that are involvedin regulation of the TP53 pathway.

SUMMARY

In one aspect, isolated synthetic duplex microRNA mimetics are provided,the synthetic duplex microRNA mimetics comprising: (i) a guide strandnucleic acid molecule consisting of a nucleotide sequence of 18 to 25nucleotides, said guide strand nucleotide sequence comprising a seedregion nucleotide sequence and a non-seed region nucleotide sequence,said seed region consisting of nucleotide positions 1 to 12 and saidnon-seed region consisting of nucleotide positions 13 to the 3′ end ofsaid guide strand, wherein position 1 of said guide strand representsthe 5′ end of said guide strand, wherein said seed region furthercomprises a consecutive nucleotide sequence of at least 6 nucleotidesthat is identical to a seed region sequence of a naturally occurringmicroRNA; and (ii) a passenger strand nucleic acid molecule consistingof a nucleotide sequence of 18 to 25 nucleotides, said passenger strandcomprising a nucleotide sequence that is essentially complementary tothe guide strand, wherein said passenger strand nucleic acid moleculehas one nucleotide sequence difference compared with the true reversecomplement sequence of the seed region of the guide strand, wherein theone nucleotide difference is located within nucleotide position 13 tothe 3′ end of the passenger strand.

In another aspect, isolated nucleic acid molecules are provided, thenucleic acid molecules comprising a guide strand nucleotide sequence of18 to 25 nucleotides, said guide strand nucleotide sequence comprising aseed region nucleotide sequence and a non-seed region nucleotidesequence, said seed region consisting essentially of nucleotidepositions 1 to 12 and said non-seed region consisting essential ofnucleotide positions 13 to the 3′ end of said guide strand, whereinposition 1 of said guide strand represents the 5′ end of said guidestrand, wherein said seed region further comprises a consecutivenucleotide sequence of at least 6 nucleotides that is identical insequence to a nucleotide sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:31 and wherein saidisolated nucleic acid molecule has at least one nucleotide sequencedifference compared to a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:29.

In another aspect, compositions are provided, the compositionscomprising at least one small interfering nucleic acid (siNA), whereinsaid siNA comprises a guide strand nucleotide sequence of 18 to 25nucleotides, said guide strand nucleotide sequence comprising a seedregion nucleotide sequence and a non-seed region nucleotide sequence,said seed region consisting essentially of nucleotide positions 1 to 12and said non-seed region consisting essentially of nucleotide positions13 to the 3′ end of said guide strand, wherein position 1 of said guidestrand represents the 5′ end of said guide strand, wherein said seedregion further comprises a consecutive nucleotide sequence of at least 6contiguous nucleotides that is identical to six contiguous nucleotideswithin a sequence selected from the group consisting of SEQ ID NO:3, SEQID NO:6, SEQ ID NO:9, and SEQ ID NO:31 and a delivery agent.

In another aspect, the invention provides a composition comprising atleast one synthetic duplex microRNA mimetic and a delivery agent, thesynthetic duplex microRNA mimetic(s) comprising:

(i) a guide strand nucleic acid molecule consisting of a nucleotidesequence of 18 to 25 nucleotides, said guide strand nucleotide sequencecomprising a seed region nucleotide sequence and a non-seed regionnucleotide sequence, said seed region consisting of nucleotide positions1 to 12 and said non-seed region consisting of nucleotide positions 13to the 3′ end of said guide strand, wherein position 1 of said guidestrand represents the 5′ end of said guide strand, wherein said seedregion further comprises a consecutive nucleotide sequence of at least 6nucleotides that is identical in sequence to a seed region of anaturally occurring microRNA; and

(ii) a passenger strand nucleic acid molecule comprising a nucleotidesequence of 18 to 25 nucleotides, said passenger strand comprising anucleotide sequence that is essentially complementary to the guidestrand, wherein said passenger strand nucleic acid molecule has onenucleotide sequence difference compared with the true reverse complementsequence of the seed region of the guide strand, wherein the onenucleotide difference is located within nucleotide position 13 to the 3′end of said passenger strand.

The isolated nucleic acid molecules and compositions of the inventionmay be used for the inhibiting cell division of a mammalian cell, suchas for the treatment of cancer in a mammalian subject.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows the RNA sequences of miR-34a, miR-34b, miR-34c, and miR-449including corresponding “seed regions”;

FIG. 2A is a histogram of cells with wildtype p53 showing the number ofcells (Y axis) with a given DNA content (measured by fluorescenceintensity, X axis);

FIG. 2B is a histogram of cells with wildtype p53 treated withdoxorubicin showing the number of cells (Y axis) with a given DNAcontent (measured by fluorescence intensity, X axis);

FIG. 2C is a histogram of cells with wildtype p53 transfected with TP53shRNA showing the number of cells (Y axis) with a given DNA content(measured by fluorescence intensity, X axis), and

FIG. 2D is a histogram of cells with wildtype p53 transfected with TP53shRNA and treated with doxorubicin showing the number of cells (Y axis)with a given DNA content (measured by fluorescence intensity, X axis),showing that disruption of TP53 ablates the G0/G1 checkpoint followingDNA damage.

DETAILED DESCRIPTION

This section presents a detailed description of the many differentaspects and embodiments that are representative of the inventionsdisclosed herein. This description is by way of several exemplaryillustrations, of varying detail and specificity. Other features andadvantages of these embodiments are apparent from the additionaldescriptions provided herein, including the different examples. Theprovided examples illustrate different components and methodology usefulin practicing various embodiments of the invention. The examples are notintended to limit the claimed invention. Based on the presentdisclosure, the ordinary skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

The present application claims priority from U.S. ProvisionalApplication Ser. No. 60/927,621 filed on May 3, 2007, which is herebyincorporated by reference.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. Practitioners are particularly directedto Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2ded., Cold Spring Harbor Press, Plainsview, N.Y. (1989), and Ausubel etal., Current Protocols in Molecular Biology (Supplement 47), John Wiley& Sons, New York (1999), for definitions and terms of the art.

It is contemplated that the use of the term “about” in the context ofthe present invention is to connote inherent problems with precisemeasurement of a specific element, characteristic, or other trait. Thus,the term “about,” as used herein in the context of the claimedinvention, simply refers to an amount or measurement that takes intoaccount single or collective calibration and other standardized errorsgenerally associated with determining that amount or measurement. Forexample, a concentration of “about” 100 mM of Tris can encompass anamount of 100 mM±0.5 mM, if 5 mM represents the collective error bars inarriving at that concentration. Thus, any measurement or amount referredto in this application can be used with the term “about” if thatmeasurement or amount is susceptible to errors associated withcalibration or measuring equipment, such as a scale, pipetteman,pipette, graduated cylinder, etc.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only, or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

As used herein, the terms “approximately” or “about” in reference to anumber are generally taken to include numbers that fall within a rangeof 5% in either direction (greater than or less than) of the numberunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value). Where rangesare stated, the endpoints are included within the range unless otherwisestated or otherwise evident from the context.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

As used herein, the term “gene” has its meaning as understood in theart. However, it will be appreciated by those of ordinary skill in theart that the term “gene” may include gene regulatory sequences (e.g.,promoters, enhancers, etc.) and/or intron sequences. It will further beappreciated that definitions of gene include references to nucleic acidsthat do not encode proteins but rather encode functional RNA moleculessuch as tRNAs. For clarity, the term gene generally refers to a portionof a nucleic acid that encodes a protein; the term may optionallyencompass regulatory sequences. This definition is not intended toexclude application of the term “gene” to non-protein coding expressionunits but rather to clarify that, in most cases, the term as used inthis document refers to a protein coding nucleic acid. In some cases,the gene includes regulatory sequences involved in transcription, ormessage production or composition. In other embodiments, the genecomprises transcribed sequences that encode for a protein, polypeptideor peptide. In keeping with the terminology described herein, an“isolated gene” may comprise transcribed nucleic acid(s), regulatorysequences, coding sequences, or the like, isolated substantially awayfrom other such sequences, such as other naturally occurring genes,regulatory sequences, polypeptide or peptide encoding sequences, etc. Inthis respect, the term “gene” is used for simplicity to refer to anucleic acid comprising a nucleotide sequence that is transcribed, andthe complement thereof.

In particular embodiments, the transcribed nucleotide sequence comprisesat least one functional protein, polypeptide and/or peptide encodingunit. As will be understood by those in the art, this functional term“gene” includes both genomic sequences, RNA or cDNA sequences, orsmaller engineered nucleic acid segments, including nucleic acidsegments of a non-transcribed part of a gene, including but not limitedto the non-transcribed promoter or enhancer regions of a gene. Smallerengineered gene nucleic acid segments may express, or may be adapted toexpress using nucleic acid manipulation technology, proteins,polypeptides, domains, peptides, fusion proteins, mutants and/or suchlike.

As used herein, the term “microRNA species”, “microRNA”, “miRNA”, or“mi-R” refers to small, non-protein coding RNA molecules that areexpressed in a diverse array of eukaryotes, including mammals. MicroRNAmolecules typically have a length in the range of from 15 to 120nucleotides, the size depending upon the specific microRNA species andthe degree of intracellular processing. Mature, fully processed miRNAsare about 15 to 30, 15 to 25, or 20 to 30 nucleotides in length, andmore often between about 16 to 24, 17 to 23, 18 to 22, 19 to 21, or 21to 24 nucleotides in length. MicroRNAs include processed sequences aswell as corresponding long primary transcripts (pri-miRNAs) andprocessed precursors (pre-miRNAs). Some microRNA molecules function inliving cells to regulate gene expression via RNA interference. Arepresentative set of microRNA species is described in the publiclyavailable miRBase sequence database as described in Griffith-Jones etal., Nucleic Acids Research 32:D109-D111 (2004) and Griffith-Jones etal., Nucleic Acids Research 34:D140-D144 (2006), accessible on the WorldWide Web at the Wellcome Trust Sanger Institute website.

As used herein, the term “microRNA family” refers to a group of microRNAspecies that share identity across at least 6 consecutive nucleotideswithin nucleotide positions 1 to 12 of the 5′ end of the microRNAmolecule, also referred to as the “seed region”, as described inBrennecke, J. et al., PloS biol 3 (3):pe85 (2005).

As used herein, the term “microRNA family member” refers to a microRNAspecies that is a member of a microRNA family.

As used herein, the term “RNA interference” or “RNAi” refers to thesilencing or decreasing of gene expression by iRNA agents (e.g., siRNAs,miRNAs, shRNAs), via the process of sequence-specific,post-transcriptional gene silencing in animals and plants, initiated byan iRNA agent that has a seed region sequence in the iRNA guide strandthat is complementary to a sequence of the silenced gene.

As used herein, the term an “iNA agent” (abbreviation for “interferingnucleic acid agent”), refers to an nucleic acid agent, for example RNA,or chemically modified RNA, which can down-regulate the expression of atarget gene. While not wishing to be bound by theory, an iNA agent mayact by one or more of a number of mechanisms, includingpost-transcriptional cleavage of a target mRNA, or pre-transcriptionalor pre-translational mechanisms. An iNA agent can include a singlestrand (ss) or can include more than one strands, e.g., it can be adouble stranded (ds) iNA agent.

As used herein, the term “single strand iRNA agent” or “ssRNA” is aniRNA agent which consists of a single molecule. It may include aduplexed region, formed by intra-strand pairing, e.g., it may be, orinclude, a hairpin or panhandle structure. The ssRNA agents of thepresent invention include transcripts that adopt stem-loop structures,such as shRNA, that are processed into a double stranded siRNA.

As used herein, the term “ds iNA agent” is a dsNA (double strandednucleic acid (NA)) agent that includes two strands that are notcovalently linked, in which interchain hybridization can form a regionof duplex structure. The dsNA agents of the present invention includesilencing dsNA molecules that are sufficiently short that they do nottrigger the interferon response in mammalian cells.

As used herein, the term “siRNA” refers to a small interfering RNA.siRNA include short interfering RNA of about 15-60, 15-50, 15-50, or15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25,or 19-25 (duplex) nucleotides in length, and preferably about 20-24 orabout 21-22 or 21-23 (duplex) nucleotides in length (e.g., eachcomplementary sequence of the double stranded siRNA is 15-60, 15-50,15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferablyabout 20-24 or about 21-22 or 21-23 nucleotides in length, preferably19-21 nucleotides in length, and the double stranded siRNA is about15-60, 15-50, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length,preferably about 20-24 or about 21-22 or 19-21 or 21-23 base pairs inlength). siRNA duplexes may comprise 3′ overhangs of about 1 to about 4nucleotides, preferably of about 2 to about 3 nucleotides and 5′phosphate termini. In some embodiments, the siRNA lacks a terminalphosphate.

Non limiting examples of siRNA molecules of the invention may include adouble-stranded polynucleotide molecule comprising self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof (alternativelyreferred to as the guide region, or guide strand when the moleculecontains two separate strands) and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof (also referred as the passenger region, or the passenger strand,when the molecule contains two separate strands). The siRNA can beassembled from two separate oligonucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (i.e., each strandcomprises a nucleotide sequence that is complementary to the nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double stranded structure, for examplewherein the double stranded region is about 18 to about 30, e.g., about18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs); theantisense strand (guide strand) comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense strand (passenger strand) comprisesnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof (e.g., about 15 to about 25 nucleotides of the siRNAmolecule are complementary to the target nucleic acid or a portionthereof). Typically, a short interfering RNA (siRNA) refers to adouble-stranded RNA molecule of about 17 to about 29 base pairs inlength, preferably from 19-21 base pairs, one strand of which iscomplementary to a target mRNA, that when added to a cell having thetarget mRNA, or produced in the cell in vivo, causes degradation of thetarget mRNA. Preferably the siRNA is perfectly complementary to thetarget mRNA. But it may have one or two mismatched base pairs.

Alternatively, the siRNA is assembled from a single oligonucleotide,where the self-complementary sense and antisense regions of the siRNAare linked by means of a nucleic acid based or non-nucleic acid-basedlinker(s). The siRNA can be a polynucleotide with a duplex, asymmetricduplex, hairpin or asymmetric hairpin secondary structure, havingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a separate target nucleic acid molecule or a portionthereof, and the sense region having nucleotide sequence correspondingto the target nucleic acid sequence or a portion thereof. The siRNA canbe a circular single-stranded polynucleotide having two or more loopstructures and a stem comprising self-complementary sense and antisenseregions, wherein the antisense region comprises nucleotide sequence thatis complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof, and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof, and wherein the circular polynucleotide can be processed eitherin vivo or in vitro to generate an active siRNA molecule capable ofmediating RNAi. The siRNA can also comprise a single strandedpolynucleotide having nucleotide sequence complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof (forexample, where such siRNA molecule does not require the presence withinthe siRNA molecule of nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof), wherein the single strandedpolynucleotide can further comprise a terminal phosphate group, such asa 5′-phosphate (see for example Martinez et al., 2002, Cell 110:563-574and Schwarz et al., 2002, Molecular Cell, 10:537-568), or5′,3′-diphosphate. In certain embodiments, the siRNA molecule of theinvention comprises separate sense and antisense sequences or regions,wherein the sense and antisense regions are covalently linked bynucleotide or non-nucleotide linkers molecules as is known in the art,or are alternately non-covalently linked by ionic interactions, hydrogenbonding, van der waals interactions, hydrophobic interactions, and/orstacking interactions. In certain embodiments, the siRNA molecules ofthe invention comprise nucleotide sequence that is complementary tonucleotide sequence of a target gene. In another embodiment, the siRNAmolecule of the invention interacts with the nucleotide sequence of atarget gene in a manner that causes inhibition of expression of thetarget gene.

As used herein, the siRNA molecules need not be limited to thosemolecules containing only RNA, but may further encompasseschemically-modified nucleotides and non-nucleotides. WO2005/078097;WO2005/0020521 and WO2003/070918 detail various chemical modificationsto RNAi molecules, wherein the contents of each reference are herebyincorporated by reference in their entirety. In certain embodiments, forexample, the short interfering nucleic acid molecules may lack2′-hydroxy (2′-OH) containing nucleotides. The siRNA can be chemicallysynthesized or may be encoded by a plasmid (e.g., transcribed assequences that automatically fold into duplexes with hairpin loops).siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNAgreater than about 25 nucleotides in length) with the E. coli RNase IIIor Dicer. These enzymes process the dsRNA into biologically active siRNA(see, e.g., Yang et al., 2002 PNAS USA 99:9942-7; Calegari et al., 2002,PNAS USA 99:14236; Byrom et al., 2003, Ambion TechNotes 10(1):4-6;Kawasaki et al., 2003, Nucleic Acids Res. 31:981-7; Knight and Bass,2001, Science 293:2269-71; and Robertson et al., 1968, J. Biol. Chem.243:82). The long dsRNA can encode for an entire gene transcript or apartial gene transcript.

As used herein, “percent modification” refers to the number ofnucleotides in each strand of the siRNA, or in the collective dsRNA,that have been modified. Thus 19% modification of the antisense strandrefers to the modification of up to 4 nucleotides/bp in a 21 nucleotidesequence (21 mer). 100% refers to a fully modified dsRNA. The extent ofchemical modification will depend upon various factors well known to oneskilled in the art. Such as, for example, target mRNA, off-targetsilencing, degree of endonuclease degradation, etc.

As used herein, the term “shRNA” or “short hairpin RNAs” refers to anRNA molecule that forms a stem-loop structure in physiologicalconditions, with a double-stranded stem of about 17 to about 29 basepairs in length, wherein one strand of the base-paired stem iscomplementary to the mRNA of a target gene. The loop of the shRNAstem-loop structure may be any suitable length that allows inactivationof the target gene in vivo. While the loop may be from 3 to 30nucleotides in length, typically it is 1-10 nucleotides in length. Thebase paired stem may be perfectly base paired or may have 1 or 2mismatched base pairs. The duplex portion may, but typically does not,contain one or more bulges consisting of one or more unpairednucleotides. The shRNA may have non-base-paired 5′ and 3′ sequencesextending from the base-paired stem. Typically, however, there is no 5′extension. The first nucleotide of the shRNA at the 5′ end is a G,because this is the first nucleotide transcribed by polymerase III. If Gis not present as the first base in the target sequence, a G may beadded before the specific target sequence. The 5′ G typically forms aportion of the base-paired stem. Typically, the 3′ end of the shRNA is apoly U segment that is a transcription termination signal and does notform a base-paired structure. As described in the application and knownto one skilled in the art, shRNAs are processed into siRNAs by theconserved cellular RNAi machinery. Thus shRNAs are precursors of siRNAsand are, in general, similarly capable of inhibiting expression of atarget mRNA transcript. For the purpose of description, in certainembodiments, the shRNA constructs of the invention target one or moremRNAs that are targeted by miR-34a, miR-34b, miR-34c or miR-449. Thestrand of the shRNA that is antisense to the target gene transcript isalso known as the “guide strand”.

As used herein, the term “microRNA responsive target site” refers to anucleic acid sequence ranging in size from about 5 to about 25nucleotides (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides) that is complementary, oressentially complementary, to at least a portion of a microRNA molecule.In some embodiments, the microRNA responsive target site comprises atleast 6 consecutive nucleotides, at least 7 consecutive nucleotides, atleast 8 consecutive nucleotides, or at least 9 nucleotides that arecomplementary to the seed region of a microRNA molecule (i.e., withinnucleotide positions 1 to 12 of the 5′ end of the microRNA molecule,referred to as the “seed region”.

The phrase “inhibiting expression of a target gene” refers to theability of an RNAi agent, such as an siRNA, to silence, reduce, orinhibit expression of a target gene. Said another way, to “inhibit”,“down-regulate”, or “reduce”, it is meant that the expression of thegene, or level of RNA molecules or equivalent RNA molecules encoding oneor more proteins or protein subunits, or activity of one or moreproteins or protein subunits, is reduced below that observed in theabsence of the RNAi agent. For example, an embodiment of the inventionproposes inhibiting, down-regulating, or reducing expression of one ormore TP53 pathway genes, by introduction of an miR-34a-like siRNAmolecule, below the level observed for that TP53 pathway gene in acontrol cell to which an mi-34a-like siRNA molecule has not beenintroduced. In another embodiment, inhibition, down-regulation, orreduction contemplates inhibition of the target mRNA below the levelobserved in the presence of, for example, an siRNA molecule withscrambled sequences or with mismatches. In yet another embodiment,inhibition, down-regulation, or reduction of gene expression with asiRNA molecule of the instant invention is greater in the presence ofthe invention siRNA, e.g., siRNA that down-regulates one or more TP53pathway gene mRNAs levels, than in its absence. In one embodiment,inhibition, down-regulation, or reduction of gene expression isassociated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation.

To examine the extent of gene silencing, a test sample (e.g., abiological sample from an organism of interest expressing the targetgene(s) or a sample of cells in culture expressing the target gene(s))is contacted with an siRNA that silences, reduces, or inhibitsexpression of the target gene(s). Expression of the target gene in thetest sample is compared to expression of the target gene in a controlsample (e.g., a biological sample from an organism of interestexpressing the target gene or a sample of cells in culture expressingthe target gene) that is not contacted with the siRNA. Control samples(i.e., samples expressing the target gene) are assigned a value of 100%.Silencing, inhibition, or reduction of expression of a target gene isachieved when the value of the test sample relative to the controlsample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, or 10%. Suitable assays include, e.g.,examination of protein or mRNA levels using techniques known to those ofskill in the art such as dot blots, northern blots, in situhybridization, ELISA, microarray hybridization, immunoprecipitation,enzyme function, as well as phenotypic assays known to those of skill inthe art.

An “effective amount” or “therapeutically effective amount” of an siRNAor an RNAi agent is an amount sufficient to produce the desired effect,e.g., inhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the siRNA or RNAiagent. Inhibition of expression of a target gene or target sequence byan siRNA or RNAi agent is achieved when the expression level of thetarget gene mRNA or protein is about 90%, 80%, 70%, 60%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, or 0% relative to the expression level of thetarget gene mRNA or protein of a control sample.

As used herein, the term “isolated” in the context of an isolatednucleic acid molecule, is one which is altered or removed from thenatural state through human intervention. For example, an RNA naturallypresent in a living animal is not “isolated.” A synthetic RNA or dsRNAor microRNA molecule partially or completely separated from thecoexisting materials of its natural state, is “isolated.” Thus, an miRNAmolecule which is deliberately delivered to or expressed in a cell isconsidered an “isolated” nucleic acid molecule.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up-regulated or down-regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

As used herein, “RNA” refers to a molecule comprising at least oneribonucleotide residue. The term “ribonucleotide” means a nucleotidewith a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety.The terms include double-stranded RNA, single-stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of an RNAiagent or internally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

As used herein, the term “complementary” refers to nucleic acidsequences that are capable of base-pairing according to the standardWatson-Crick complementary rules. That is, the larger purines will basepair with the smaller pyrimidines to form combinations of guanine pairedwith cytosine (G:C) and adenine paired with either thymine (A:T) in thecase of DNA, or adenine paired with uracil (A:U) in the case of RNA.

As used herein, the term “essentially complementary” with reference tomicroRNA target sequences refers to microRNA target nucleic acidsequences that are longer than 8 nucleotides that are complementary (anexact match) to at least 8 consecutive nucleotides of the 5′ portion ofa microRNA molecule from nucleotide positions 1 to 12, (also referred toas the “seed region”), and are at least 65% complementary (such as atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 96% identical) across the remainder of themicroRNA target nucleic acid sequence as compared to a naturallyoccurring miR-34 family member. The comparison of sequences anddetermination of percent identity and similarity between two sequencescan be accomplished using a mathematical algorithm of Karlin andAltschul (1990, PNAS 87:2264-2268), modified as in Karlin and Altschul(1993, PNAS 90:5873-5877). Such an algorithm is incorporated into theNBLAST and XBLAST programs of Altschul et al. (1990 J. Mol. Biol.215:403-410).

As used herein, the term “gene” encompasses the meaning known to one ofskill in the art, i.e., a nucleic acid (e.g., DNA or RNA) sequence thatcomprises coding sequences necessary for the production of an RNA and/ora polypeptide, or its precursor as well as noncoding sequences(untranslated regions) surrounding the 5′ and 3′ ends of the codingsequences. The term “gene” encompasses both cDNA and genomic forms of agene. The term “gene” also encompasses nucleic acid sequences thatcomprise microRNAs and other non-protein encoding sequences, including,for example, transfer RNAs, ribosomal RNAs, etc. A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, antigenic presentation) of the polypeptide are retained.The sequences which are located 5′ of the coding region and which arepresent on the mRNA are referred to as 5′ untranslated sequences(“5′UTR”). The sequences which are located 3′ or downstream of thecoding region and which are present on the mRNA are referred to as 3′untranslated sequences, or (“3′UTR”).

The term “gene expression”, as used herein, refers to the process oftranscription and translation of a gene to produce a gene product, be itRNA or protein. Thus, modulation of gene expression may occur at any oneor more of many levels, including transcription, post-transcriptionalprocessing, translation, post-translational modification, and the like.

As used herein, the term “expression cassette” refers to a nucleic acidmolecule which comprises at least one nucleic acid sequence that is tobe expressed, along with its transcription and translational controlsequences. The expression cassette typically includes restriction sitesengineered to be present at the 5′ and 3′ ends such that the cassettecan be easily inserted, removed, or replaced in a gene delivery vector.Changing the cassette will cause the gene delivery vector into which itis incorporated to direct the expression of a different sequence.

As used herein, the term “phenotype” encompasses the meaning known toone of skill in the art, including modulation of the expression of oneor more genes, as measured by gene expression analysis or proteinexpression analysis.

As used herein, the term “proliferative disease” or “cancer” as usedherein refers to any disease, condition, trait, genotype or phenotypecharacterized by unregulated cell growth or replication as is known inthe art; including leukemias, for example, acute myelogenous leukemia(AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia(ALL), and chronic lymphocytic leukemia; AIDS related cancers such asKaposi's sarcoma; breast cancers; bone cancers such as osteosarcoma,chondrosarcomas, Ewing's sarcoma, fibrosarcomas, giant cell tumors,adamantinomas, and chordomas; brain cancers such as meningiomas,glioblastomas, lower-grade astrocytomas, oligodendrocytomas, pituitarytumors, schwannomas, and metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease-related gene expression in a cell or tissue, aloneor in combination with other therapies.

As used herein, the term “source of biological knowledge” refers toinformation that describes the function (e.g., at molecular, cellular,and system levels), structure, pathological roles, toxicologicalimplications, etc., of a multiplicity of genes. Various sources ofbiological knowledge can be used for the methods of the invention,including databases and information collected from public sources suchas Locuslink, Unigene, SwissTrEMBL, etc., and organized into arelational database following the concept of the central dogma ofmolecular biology. In some embodiments, the annotation systems used bythe Gene Ontology (GO) Consortium or similar systems are employed. GO isa dynamic controlled vocabulary for molecular biology which can beapplied to all organisms. As knowledge of gene function is accumulatingand changing, it is developed and maintained by the Gene Ontology™Consortium (Gene Ontology: tool for the unification of biology. The GeneOntology Consortium (2000), Nature Genet. 25:25-29)).

As used herein, the term to “inhibit the proliferation of a mammaliancell” means to kill the cell, or permanently or temporarily arrest thegrowth of the cell. Inhibition of a mammalian cell can be inferred ifthe number of such cells, either in an in vitro culture vessel, or in asubject, remains constant or decreases after administration of thecompositions of the invention. An inhibition of tumor cell proliferationcan also be inferred if the absolute number of such cells increases, butthe rate of tumor growth decreases.

As used herein, the terms “measuring expression levels,” “obtaining anexpression level” and the like, include methods that quantify a geneexpression level of, for example, a transcript of a gene, includingmicroRNA (miRNA) or a protein encoded by a gene, as well as methods thatdetermine whether a gene of interest is expressed at all. Thus, an assaywhich provides a “yes” or “no” result without necessarily providingquantification, of an amount of expression is an assay that “measuresexpression” as that term is used herein. Alternatively, a measured orobtained expression level may be expressed as any quantitative value,for example, a fold-change in expression, up or down, relative to acontrol gene or relative to the same gene in another sample, or a logratio of expression, or any visual representation thereof, such as, forexample, a “heatmap” where a color intensity is representative of theamount of gene expression detected. Exemplary methods for detecting thelevel of expression of a gene include, but are not limited to, Northernblotting, dot or slot blots, reporter gene matrix (see for example, U.S.Pat. No. 5,569,588) nuclease protection, RT-PCR, microarray profiling,differential display, 2D gel electrophoresis, SELDI-TOF, ICAT, enzymeassay, antibody assay, and the like.

As used herein “miR-34 family” refers to miR-34a, miR34b, miR34 c, andmiR-449.

As used herein, “miR-34” refers to one or more of miR-34a, miR-34b andmiR34c.

As used herein, “miR-34a” refers to SEQ ID NO:1 and precursor RNAssequences thereof, an example of which is SEQ ID NO:2.

As used herein, “miR-34a seed region” refers to SEQ ID NO:3

As used herein, “miR-34b” refers to SEQ ID NO:4 and precursor RNAssequences thereof, an example of which is SEQ ID NO:5.

As used herein, “miR-34b seed region” refers to SEQ ID NO:6

As used herein, “miR-34c” refers to SEQ ID NO:7 and precursor RNAssequences thereof, an example of which is SEQ ID NO:8.

As used herein, “miR-34c seed region” refers to SEQ ID NO:9

As used herein “miR-449” refers to SEQ ID NO:29 and precursor RNAssequences thereof, an example of which is SEQ ID NO:30.

As used herein, “miR-449 seed region” refers to SEQ ID NO:31.

As used herein, an “isolated nucleic acid” is a nucleic acid moleculethat exists in a physical form that is non-identical to any nucleic acidmolecule of identical sequence as found in nature; “isolated” does notrequire, although it does not prohibit, that the nucleic acid sodescribed has itself been physically removed from its nativeenvironment. For example, a nucleic acid can be said to be “isolated”when it includes nucleotides and/or internucleoside bonds not found innature. When instead composed of natural nucleosides in phosphodiesterlinkage, a nucleic acid can be said to be “isolated” when it exists at apurity not found in nature, where purity can be adjudged with respect tothe presence of nucleic acids of other sequence, with respect to thepresence of proteins, with respect to the presence of lipids, or withrespect to the presence of any other component of a biological cell, orwhen the nucleic acid lacks sequence that flanks an otherwise identicalsequence in an organism's genome, or when the nucleic acid possessessequence not identically present in nature. As so defined, “isolatednucleic acid” includes nucleic acids integrated into a host cellchromosome at a heterologous site, recombinant fusions of a nativefragment to a heterologous sequence, recombinant vectors present asepisomes or as integrated into a host cell chromosome.

The terms “over-expression”, “over-expresses”, “over-expressing” and thelike, refer to the state of altering a subject such that expression ofone or more genes in said subject is significantly higher, as determinedusing one or more statistical tests, than the level of expression ofsaid gene or genes in the same unaltered subject or an analogousunaltered subject.

As used herein, a “purified nucleic acid” represents at least 10% of thetotal nucleic acid present in a sample or preparation. In preferredembodiments, the purified nucleic acid represents at least about 50%, atleast about 75%, or at least about 95% of the total nucleic acid in anisolated nucleic acid sample or preparation. Reference to “purifiednucleic acid” does not require that the nucleic acid has undergone anypurification and may include, for example, chemically synthesizednucleic acid that has not been purified.

As used herein, “specific binding” refers to the ability of twomolecular species concurrently present in a heterogeneous(inhomogeneous) sample to bind to one another in preference to bindingto other molecular species in the sample. Typically, a specific bindinginteraction will discriminate over adventitious binding interactions inthe reaction by at least 2-fold, more typically by at least 10-fold,often at least 100-fold; when used to detect analyte, specific bindingis sufficiently discriminatory when determinative of the presence of theanalyte in a heterogeneous (inhomogeneous) sample. Typically, theaffinity or avidity of a specific binding reaction is least about 1 μM.

As used herein, “subject”, as refers to an organism or to a cell sample,tissue sample or organ sample derived therefrom, including, for example,cultured cell lines, biopsy, blood sample of fluid sample containing acell. For example, an organism may be an animal, including but notlimited to, an animal such as a cow, a pig, a mouse, a rat, a chicken, acat, a dog, etc., and is usually a mammal, such as a human.

As used herein, “TP53 pathway” refers to proteins, and theircorresponding genes, that function both upstream and downstream of TP53,including, for example, proteins that are involved in or required forperception of DNA damage, modulation of TP53 activity, cell cyclearrest, and apoptosis. TP53 pathway includes, but is not limited to, thegenes, and proteins encoded thereby, listed in Table 1 (see alsoVogelstein, et al., 2000, Nature 408:307-310; Woods and Vousden, 2001,Experimental Cell Research 264:56-66; El-Deiry, 1998, Semin. CancerBiology 8:345-357; and Prives and Hall, 1999, J. Pathol. 1999187:112-126).

TABLE 1 TP53 Pathway Genes GeneBank Symbol Description GO Term NM_002954RPS27A Ribosomal Intracellular; Protein protein S27a biosynthesis;Structural constituent of ribosome; Ribosome; NM_012138 AATF ApoptosisNucleus; Anti-apoptosis; antagonizing Transcription factor transcriptionactivity; factor NM_001160 APAF1 Apoptotic ATP binding; Proteinpeptidase binding; Regulation of activating factor apoptosis; Cytosol;Intracellular; Caspase activation via cyto- chrome c; Neurogenesis;Caspase activator activity; NM_000051 ATM Ataxia Transferase activity;telangiectasia Signal transduction; mutated DNA binding; Regula-(includes tion of transcription, complementation DNA-dependent; groupsA, C and Nucleus; Protein serine/ D) threonine kinase activity; Negativeregulation of cell cycle; Transcription factor activity; Intra-cellular; DNA repair; Phosphotransferase activity, alcohol group asacceptor; Meiotic recombination; NM_001184 ATR Ataxia Development;Protein telangiectasia kinase activity; Cell and Rad3 related cycle;Cell cycle checkpoint; DNA repair; NM_004323 BAG1 BCL2-associatedReceptor signaling athanogene protein activity; Cyto- plasm; Apoptosis;Anti- apoptosis; Cell surface receptor linked signal transduction;Protein folding; Unfolded protein binding; NM_001702 BAI1 Brain-specificCell adhesion; Signal angiogenesis transduction; Protein inhibitor 1binding; Negative regulation of cell pro- liferation; Integral to plasmamembrane; Axonogenesis; Inter- cellular junction; G- protein coupledreceptor activity; Neuropeptide signaling pathway; Peripheral nervoussystem development; Brain-specific angiogenesis inhibitor activity;NM_001188 BAK1 BCL2- Integral to membrane; antagonist/killer Apoptoticmitochondrial 1 changes; Induction of apoptosis; Regulation ofapoptosis; NM_004656 BAP1 BRCA1 Nucleus; Negative associated regulationof cell protein-1 proliferation; Ubiquitin- (ubiquitin dependent proteincarboxy-terminal catabolism; Peptidase hydrolase) activity; Proteinmodification; Ubiquitin thiolesterase activity; NM_004324 BAXBCL2-associated Integral to membrane; X protein Negative regulation ofcell cycle; Apoptotic mitochondrial changes; Induction of apoptosis;Regulation of apoptosis; Molecular_function unknown; Apoptosis; Germcell development; Induction of apoptosis by extracellular signals;Negative regulation of survival gene product activity; NM_000633 BCL2B-cell Integral to membrane; CLL/lymphoma Protein binding; Cell 2 growthand/or mainte- nance; Regulation of apoptosis; Anti- apoptosis; Humoralimmune response; Negative regulation of cell proliferation; Regu- lationof cell cycle; Mito- chondrial outer mem- brane; Mitochondrion;NM_004049 BCL2A1 BCL2-related Regulation of apoptosis; protein A1Anti-apoptosis; Intra- cellular; NM_001196 BID BH3 interacting Apoptoticmitochondrial domain death changes; Regulation of agonist apoptosis;Mitochon- drion; Death receptor binding; Induction of apoptosis viadeath domain receptors; Cytosol; Membrane fraction; NM_001168 BIRC5Baculoviral IAP Microtubule binding; repeat-containing Apoptosis; Anti-5 (survivin) apoptosis; Zinc ion binding; Intracellular; Caspaseinhibitor activity; G2/M transition of mitotic cell cycle; Cysteineprotease inhibitor activity; Protease inhibitor activity; Spindlemicrotubule; NM_004052 BNIP3 BCL2/adenovirus Integral to membrane; E1B19 kDa Protein binding; Apop- interacting tosis; Anti-apoptosis; protein3 Mitochondrion; NM_007294 BRCA1 Breast cancer 1, Nucleus; Proteinbinding; early onset Negative regulation of cell cycle; Regulation ofapoptosis; Zinc ion bind- ing; Ubiquitin-protein ligase activity;Protein ubiquitination; Ubiquitin ligase complex; Regula- tion oftranscription from Pol II promoter; Tran- scriptional activatoractivity; Intracellular; Extracellular space; Transcription factorcomplex; Transcription coactivator activity; Damaged DNA binding;Tubulin binding; DNA damage response, signal transduction by p53 classmediator resulting in transcription of p21 class mediator; Negativeregulation of centriole replication; Positive regulation of DNA repair;Regulation of cell proliferation; Regulation of transcription from PolIII promoter; Gamma- tubulin ring complex; NM_000059 BRCA2 Breast cancer2, Nucleic acid binding; early onset Nucleus; Protein binding;Regulation of cell cycle; Extracellular space; Transcription coactivatoractivity; DNA repair; Single-stranded DNA binding; Chromatin remodeling;Double- strand break repair via homologous recombina- tion;Establishment and/or maintenance of chromatin architecture; Regulationof transcrip- tion; Secretory granule; Mitotic checkpoint; Regulation ofS phase of mitotic cell cycle; NM_006763 BTG2 BTG family, Regulation oftranscrip- member 2 tion, DNA-dependent; Negative regulation of cellproliferation; Transcription factor activity; DNA repair; NM_032982CASP2 Caspase 2, Hydrolase activity; apoptosis-related Proteolysis andcysteine peptidolysis; Protein peptidase (neural binding; Regulation ofprecursor cell apoptosis; Caspase expressed, activity; Cysteine-typedevelopmentally peptidase activity; down-regulated Apoptotic program; 2)Enzyme binding; Intracellular; NM_001229 CASP9 Caspase 9, Proteolysisand apoptosis-related peptidolysis; Protein cysteine binding; Regulationof peptidase apoptosis; Caspase activity; Apoptotic program;Intracellular; Caspase activation via cytochrome c; Enzyme activatoractivity; NM_057735 CCNE2 Cyclin E2 Nucleus; Regulation of cell cycle;Regulation of cyclin dependent protein kinase activity; Cell cyclecheckpoint; NM_004354 CCNG2 Cyclin G2 Cell cycle; Cell cycle checkpoint;Mitosis; NM_001239 CCNH Cyclin H Regulation of transcrip- tion,DNA-dependent; Nucleus; Cell cycle; Regulation of cyclin dependentprotein kinase activity; DNA repair; NM_001786 CDC2 Cell division ATPbinding; cycle 2, G1 to S Transferase activity; and G2 to M Proteinamino acid phosphorylation; Nucleus; Protein serine/threonine kinaseactivity; Protein-tyrosine kinase activity; Cyclin- dependent proteinkinase activity; Mitosis; Traversing start control point of mitotic cellcycle; NM_001789 CDC25A Cell division Hydrolase activity; Cell cycle 25Aproliferation; Intracellu- lar; Regulation of cyclin dependent proteinkinase activity; Mitosis; Protein amino acid dephosphor- ylation;Protein tyrosine phosphatase activity; M phase of mitotic cell cycle;NM_001790 CDC25C Cell division Hydrolase activity; Cell cycle 25Cproliferation; Nucleus; Regulation of cyclin dependent protein kinaseactivity; Protein amino acid dephosphorylation; Protein tyrosinephospha- tase activity; Regulation of mitosis; Traversing start controlpoint of mitotic cell cycle; NM_000075 CDK4 Cyclin-dependent ATPbinding; kinase 4 Transferase activity; Protein amino acidphosphorylation; Regulation of cell cycle; Cyclin-dependent proteinkinase activity; Protein kinase activity; G1/S transition of mitoticcell cycle; NM_001799 CDK7 Cyclin-dependent ATP binding; kinase 7 (MO15Transferase activity; homolog, Protein amino acid Xenopus laevis,phosphorylation; cdk-activating Regulation of transcrip- kinase) tion,DNA-dependent; Nucleus; Cyclin- dependent protein kinase activity;Regulation of cyclin dependent protein kinase activity; DNA repair;Transcription initiation from Pol II promoter; NM_000389 CDKN1ACyclin-dependent Nucleus; Negative kinase inhibitor regulation of cell1A (p21, Cip1) proliferation; Cell cycle arrest; Protein kinaseactivity; Cyclin- dependent protein kinase inhibitor activity;Regulation of cyclin dependent protein kinase activity; Kinase activity;Induction of apoptosis by intracellular signals; NM_000077 CDKN2ACyclin-dependent Nucleus; Negative kinase inhibitor regulation of cellcycle; 2A (melanoma, Negative regulation of p16, inhibits cellproliferation; Cell CDK4) cycle arrest; Cell cycle; Cyclin-dependentprotein kinase inhibitor activity; Regulation of cyclin dependentprotein kinase activity; Kinase activity; Cell cycle checkpoint;NM_001274 CHEK1 CHK1 ATP binding; checkpoint Transferase activity;homolog Protein amino acid (S. pombe) phosphorylation; Nucleus; Proteinserine/threonine kinase activity; Negative regulation of cellproliferation; Cell cycle; Regulation of cyclin dependent protein kinaseactivity; Meiotic recom- bination; DNA damage checkpoint; Response toDNA damage stimulus; Gametogenesis; Condensed nuclear chromosome;NM_007194 CHEK2 CHK2 ATP binding; Trans- checkpoint ferase activity;Protein homolog amino acid phos- (S. pombe) phorylation; Nucleus;Protein serine/threonine kinase activity; Cell growth and/or maint-enance; Protein kinase activity; Cell cycle; DNA damage checkpoint;Response to DNA damage stimulus; NM_004804 WDR39 WD repeat Nucleus;Positive domain 39 regulation of cell prolif- eration; Regulation oftranscription from Pol II promoter; NM_001300 KLF6 Kruppel-like Nucleicacid binding; factor 6 DNA binding; Regula- tion of transcription,DNA-dependent; Nucleus; Zinc ion binding; Transcriptional activatoractivity; Cell growth; B-cell differentiation; NM_003805 CRADD CASP2 andSignal transduction; RIPK1 domain Protein binding; containing Regulationof apoptosis; adaptor with Induction of apoptosis death domain via deathdomain receptors; Intracellular; NM_001554 CYR61 Cysteine-rich, Celladhesion; Cell angiogenic proliferation; Regulation inducer, 61 of cellgrowth; Extra- cellular; Heparin binding; Chemotaxis; Insulin-likegrowth factor binding; Morphogenesis; NM_004938 DAPK1 Death-associatedATP binding; Trans- protein kinase 1 ferase activity; Protein amino acidphos- phorylation; Protein kinase cascade; Signal transduction; Proteinserine/threonine kinase activity; Apoptosis; Induction of apoptosis byextracellular signals; Calmodulin binding; Actin cytoskeleton; Calcium-and calmodulin-dependent protein kinase activity; Calmodulin-dependentprotein kinase I activity; NM_001350 DAXX Death-associated Calcium ionbinding; protein 6 Regulation of transcrip- tion, DNA-dependent;Nucleus; Apoptosis; NM_005225 E2F1 E2F transcription Regulation oftranscrip- factor 1 tion, DNA-dependent; Nucleus; Apoptosis; Regulationof cell cycle; Transcription factor activity; Negative regulation oftranscrip- tion from Pol II promot- er; G1 phase of mitotic cell cycle;Transcription corepressor activity; Transcription factor complex;NM_001949 E2F3 E2F transcription Regulation of transcrip- factor 3 tion,DNA-dependent; Nucleus; Protein binding; Regulation of cell cycle;Transcription factor activity; Transcription factor complex;Transcription initiation from Pol II promoter; NM_004879 EI24 EtoposideInduction of apoptosis; induced 2.4 mRNA NM_000125 ESR1 Estrogen Signaltransduction; receptor 1 DNA binding; Regula- tion of transcription,DNA-dependent; Nucleus; Transcription factor activity; Receptoractivity; Membrane; Steroid hormone receptor activity; Cell growth;Nitric-oxide synthase regulator activity; Steroid binding; Estrogenreceptor activity; Estrogen receptor signaling path- way; Negativeregulation of mitosis; Chromatin remodeling complex; NM_003824 FADD Fas(TNFRSF6)- Protein binding; Cyto- associated via plasm; Regulation ofdeath domain apoptosis; Death receptor binding; Induction of apoptosisvia death domain receptors; Signal transducer activity; Cell surfacereceptor linked signal transduction; Positive regulation of I- kappaBkinase/NF- kappaB cascade; Antimicrobial humoral response (sensuVertebrata); NM_007051 FAF1 Fas (TNFRSF6) Nucleus; associated factorMolecular_function 1 unknown; Apoptosis; NM_001455 FOXO3A Forkhead boxRegulation of transcrip- O3A tion, DNA-dependent; Nucleus; Cytoplasm;Cell growth and/or maintenance; Induction of apoptosis; Apoptosis;Transcription factor activity; Transcription from Pol II promoter;NM_004958 FRAP1 FK506 binding Transferase activity; protein 12-Regulation of cell cycle; rapamycin DNA recombination; associated DNArepair; Inositol or protein 1 phosphatidylinositol kinase activity;Phosphoinositide 3-kinase complex; NM_001924 GADD45A Growth arrestNucleus; Apoptosis; Cell and DNA- cycle arrest; Regulation damage- ofcyclin dependent inducible, alpha protein kinase activity; DNA repair;Protein biosynthesis; Structural constituent of ribosome; Ribosome;NM_005255 GAK Cyclin G ATP binding; Trans- associated kinase feraseactivity; Protein amino acid phos- phorylation; Nucleus; Proteinserine/threonine kinase activity; Regulation of cell cycle; Kinaseactivity; Endoplasmic reticulum; NM_002048 GAS1 Growth arrest-Molecular_function specific 1 unknown; Negative regulation of cellproliferation; Cell cycle arrest; Extrinsic to plasma membrane,GPI-anchored; Negative regulation of S phase of mitotic cell cycle;NM_002066 GML GPI anchored Plasma membrane; molecule like Apoptosis;Negative protein regulation of cell proliferation; Regulation of cellcycle; Extrinsic to membrane; DNA damage response, signal trans- ductionby p53 class mediator resulting in cell cycle arrest; NM_016426 GTSE1G-2 and S-phase Molecular_function expressed 1 unknown; G2 phase ofmitotic cell cycle; Microtubule-based process; DNA damage response,signal trans- duction by p53 class mediator resulting in cell cyclearrest; Cytoplasmic microtubule; NM_004964 HDAC1 Histone Hydrolaseactivity; deacetylase 1 Regulation of transcription, DNA- dependent;Nucleus; Cytoplasm; Anti- apoptosis; Transcription factor activity;Transcription factor binding; Histone deacetylase activity; Chromatinmodification; Histone deacetylation; Histone deacetylase complex;NM_000189 HK2 Hexokinase 2 ATP binding; Transferase activity; Regulationof cell cycle; Mitochondrial outer membrane; Membrane; Glycolysis;Kinase activity; Hexokinase activity; NM_002176 IFNB1 Interferon, betaExtracellular; Negative 1, fibroblast regulation of cell proliferation;Cell surface receptor linked signal transduction; Response to virus;Caspase activation; B-cell proliferation; Defense response; Naturalkiller cell activation; Positive regulation of innate immune response;Interferon-alpha/beta receptor binding; Anti- inflammatory response;Negative regulation of virion penetration; Regulation of MHC class Ibiosynthesis; NM_000875 IGF1R Insulin-like ATP binding; Transferasegrowth factor 1 activity; Protein amino receptor acid phosphorylation;Integral to membrane; Signal transduction; Protein binding; Anti-apoptosis; Regulation of cell cycle; Positive regulation of cellproliferation; Receptor activity; Epidermal growth factor receptoractivity; Insulin-like growth factor receptor activity; Insulin receptorsignaling pathway; NM_000600 IL6 Interleukin 6 Humoral immune(interferon, beta response; Negative 2) regulation of cellproliferation; Positive regulation of cell proliferation; Cell surfacereceptor linked signal transduction; Extracellular space; Acute-phaseresponse; Cell-cell signaling; Cytokine activity; Interleukin-6 receptorbinding; NM_002228 JUN V-jun sarcoma Regulation of virus 17transcription, DNA- oncogene dependent; Cell growth homolog (avian)and/or maintenance; Transcription factor activity; RNA polymerase IItranscription factor activity; Nuclear chromosome; NM_004985 KRASV-Ki-ras2 GTP binding; GTPase Kirsten rat activity; Small GTPase sarcomaviral mediated signal oncogene transduction; Cell growth homolog and/ormaintenance; Regulation of cell cycle; NM_018494 LRDD Leucine-richSignal transduction; repeats and death Protein binding; Death domainreceptor binding; containing NM_021960 MCL1 Myeloid cell Integral tomembrane; leukemia Protein binding; sequence Cytoplasm; Regulation of 1(BCL2-related) apoptosis; Anti-apoptosis; Mitochondrial outer membrane;Apoptotic program; Cell differentiation; Protein channel activity;Protein heterodimerization activity; Cell fate determination; Cellhomeostasis; NM_002392 MDM2 Mdm2, Nucleus; Protein binding; transformed3T3 Cell growth and/or cell double maintenance; Protein minute 2, p53complex assembly; binding protein Negative regulation of cell (mouse)proliferation; Regulation of cell cycle; Zinc ion binding; Negativeregulation of transcription from Pol II promoter; Ligase activity;Ubiquitin- protein ligase activity; Protein ubiquitination; Ubiquitinligase complex; Negative regulator of basal transcription activity;Regulation of protein catabolism; Nucleolus; Nucleoplasm; NM_002393 MDM4Mdm4, Nucleus; Protein binding; transformed 3T3 Protein complexassembly; cell double Negative regulation of cell minute 4, p53proliferation; Zinc ion binding protein binding; Negative (mouse)regulation of transcription from Pol II promoter; Ubiquitin-proteinligase activity; Protein ubiquitination; Ubiquitin ligase complex;Protein stabilization; Negative regulation of protein catabolism;NM_000251 MSH2 MutS homolog 2, ATP binding; Nucleus; colon cancer,Negative regulation of cell nonpolyposis cycle; Mismatch repair; type 1(E. coli) Damaged DNA binding; Postreplication repair; NM_002467 MYCV-myc Cell proliferation; Nucleus; myelocyto- Transcription factormatosis viral activity; Regulation of oncogene transcription from Pol IIhomolog (avian) promoter; Cell cycle arrest; Iron ion homeostasis;NM_002478 MYOD1 Myogenic Protein amino acid differentiation 1phosphorylation; DNA binding; RNA polymerase II transcription factoractivity, enhancer binding; Regulation of transcrip- tion,DNA-dependent; Nucleus; Regulation of transcription from Pol IIpromoter; Muscle development; Transcrip- tion coactivator activity; Celldifferentiation; Myogenesis; NM_006096 NDRG1 N-myc Nucleus; Celldownstream differentiation; Catalytic regulated gene 1 activity;Response to metal ion; NM_000267 NF1 Neurofibromin 1 Cytoplasm; Cellgrowth (neurofibro- and/or maintenance; matosis, von Negative regulationof cell Recklinghausen cycle; Negative regulation disease, Watson ofcell proliferation; RAS disease) protein signal transduction; Ras GTPaseactivator activity; Enzyme inhibitor activity; NM_003998 NFKB1 Nuclearfactor of Signal transduction; kappa light Regulation of tran-polypeptide gene scription, DNA-dependent; enhancer in B- Nucleus;Protein binding; cells 1 (p105) Cytoplasm; Apoptosis; Anti-apoptosis;Transcrip- tion factor activity; Inflammatory response; Transcriptionfrom Pol II promoter; Response to pathogenic bacteria; Antibacterialhumoral response (sensu Vertebrata); NM_022112 P53AIP1 P53-regulatedMolecular_function apoptosis-induc- unknown; Apoptosis; ing protein 1Mitochondrion; NM_003884 PCAF P300/CBP- Transferase activity; associatedfactor Regulation of tran- scription, DNA-dependent; Nucleus; Negativeregulation of cell proliferation; Cell cycle arrest; Cell cycle;Chromatin remodeling; Transcription cofactor activity; N-acetyltransferase activity; Histone acetyltransferase activity; Proteinamino acid acetylation; NM_020418 PCBP4 Poly(rC) binding Nucleic acidbinding; protein 4 DNA binding; Nucleus; DNA damage response, signaltransduction resulting in induction of apoptosis; Cell cycle arrest; RNAbinding; Ribonucleoprotein complex; DNA damage response, signaltransduction by p53 class mediator resulting in cell cycle arrest; MRNAmetabolism; NM_002634 PHB Prohibitin Cell growth and/or maintenance;Negative regulation of cell proliferation; Regulation of cell cycle;Transcriptional activator activity; Nucleoplasm; DNA metabolism; Histonedeacetylation; Mitochondrial inner membrane; Transcriptional repressoractivity; Negative regulation of transcription; NM_002656 PLAGL1Pleiomorphic Nucleic acid binding; adenoma gene- DNA binding; Regulationlike 1 of transcription, DNA- dependent; Nucleus; Induction ofapoptosis; Zinc ion binding; Cell cycle arrest; NM_005030 PLK1 Polo-likekinase ATP binding; Transferase 1 (Drosophila) activity; Protein aminoacid phosphorylation; Nucleus; Protein serine/threonine kinase activity;Regulation of cell cycle; Mitosis; NM_033238 PML Promyelocytic Nucleicacid binding; leukemia Regulation of transcrip- tion, DNA-dependent;Nucleus; Cell growth and/or maintenance; Transcription factor activity;Zinc ion binding; Ubiquitin-protein ligase activity; Proteinubiquitination; Ubiquitin ligase complex; Transcription cofactoractivity; NM_000304 PMP22 Peripheral Negative regulation of cell myelinprotein 22 proliferation; Membrane fraction; Integral to plasmamembrane; Perception of sound; Synaptic transmission; Peripheral nervoussystem development; Mechanosensory behavior; NM_003620 PPM1D ProteinHydrolase activity; phosphatase 1D Nucleus; Negative magnesium-regulation of cell dependent, delta proliferation; Regulation of isoformcell cycle; Protein amino acid dephosphorylation; Response to radiation;Magnesium ion binding; Manganese ion binding; Protein phosphatase type2C activity; Protein serine/threonine phosphatase complex; NM_015316PPP1R13B Protein phosphatase 1, regulatory (inhibitor) subunit 13BNM_032595 PPP1R9B Protein Protein binding; phosphatase 1, Cytoplasm;Cell cycle regulatory arrest; Nucleoplasm; subunit 9B, Negativeregulation of cell spinophilin growth; Regulation of cell proliferation;Protein phosphatase inhibitor activity; RNA splicing; Regulation of exitfrom mitosis; Protein phosphatase 1 binding; Interpretation of externalsignals that regulate cell growth; Protein phosphatase type 1 complex;NM_002737 PRKCA Protein kinase C, ATP binding; Transferase alphaactivity; Protein amino acid phosphorylation; Calcium ion binding;Diacylglycerol binding; Intracellular signaling cascade; Induction ofapoptosis by extracellular signals; Regulation of cell cycle; Membranefraction; Cell surface receptor linked signal transduction; Proteinkinase C activity; NM_006257 PRKCQ Protein kinase C, ATP binding;Transferase theta activity; Protein amino acid phosphorylation;Regulation of cell growth; Diacylglycerol binding; Proteinserine/threonine kinase activity; Intracellular signaling cascade;Intracellular; NM_000314 PTEN Phosphatase and Hydrolase activity; Celltensin homolog cycle; Protein amino acid (mutated in dephosphorylation;Protein multiple tyrosine phosphatase advanced cancers activity;Protein 1) tyrosine/serine/threonine phosphatase activity;Phosphatidylinositol-3,4,5- trisphosphate 3- phosphatase activity;Negative regulation of progression though cell cycle; NM_004219 PTTG1Pituitary tumor- Nucleus; Protein binding; transforming 1 Cytoplasm;Cell growth and/or maintenance; Transcription factor activity;Transcription from Pol II promoter; DNA repair; Spermatogenesis; DNAmetabolism; Mitosis; Cysteine protease inhibitor activity; DNAreplication and chromosome cycle; Chromosome segregation; NM_013258PYCARD PYD and CARD Signal transduction; domain Protein binding;containing Cytoplasm; Negative regulation of cell cycle; Induction ofapoptosis; Regulation of apoptosis; Caspase activator activity; Caspaseactivation; NM_006663 PPP1R13L Protein Regulation of phosphatase 1,transcription, DNA- regulatory dependent; Nucleus; (inhibitor)Apoptosis; subunit 13 like NM_000321 RB1 Retinoblastoma 1 Regulation of(including transcription, DNA- osteosarcoma) dependent; Nucleus;Negative regulation of cell cycle; Transcription factor activity;Negative regulation of transcription from Pol II promoter; Chromatin;Cell cycle checkpoint; NM_021975 RELA V-rel Regulation of transcription,reticuloendo- DNA-dependent; Nucleus; theliosis viral Protein binding;Anti- oncogene apoptosis; Transcription homolog A, factor activity;Signal nuclear factor of transducer activity; kappa light Positiveregulation of I- polypeptide gene kappaB kinase/NF-kappaB enhancer in B-cascade; Transcription cells 3, p65 from Pol II promoter; (avian)Transcription factor complex; Response to toxin; NM_019845 RPRM Reprimo,TP53 Cytoplasm; Cell cycle dependent G2 arrest; arrest mediatorcandidate NM_052863 SCGB3A1 Secretoglobin, Extracellular; Negativefamily 3A, regulation of cell growth; member 1 Regulation of cellproliferation; Cytokine activity; NM_014454 SESN1 Sestrin 1 Nucleus;Negative regulation of cell proliferation; Cell cycle arrest; Responseto DNA damage stimulus; NM_031459 SESN2 Sestrin 2 Nucleus; Cell cyclearrest; NM_144665 SESN3 Sestrin 3 Nucleus; Cell cycle arrest; NM_006142SFN Stratifin Cell proliferation; Signal transduction; Cytoplasm;Regulation of cell cycle; Extracellular space; Protein domain specificbinding; Protein kinase C inhibitor activity; Negative regulation ofprotein kinase activity; NM_003029 SHC1 SHC (Src Plasma membrane;homology 2 Regulation of cell growth; domain Intracellular signalingcontaining) cascade; Positive transforming regulation of cell protein 1proliferation; Activation of MAPK; Phospholipid binding; Transmembranereceptor protein tyrosine kinase adaptor protein activity; Positiveregulation of mitosis; Regulation of epidermal growth factor receptoractivity; NM_003031 SIAH1 Seven in absentia Nucleus; Apoptosis; Zinchomolog 1 ion binding; Ligase (Drosophila) activity; Development; Cellcycle; Spermatogenesis; Ubiquitin-dependent protein catabolism;Ubiquitin cycle; NM_012238 SIRT1 Sirtuin (silent Hydrolase activity; DNAmating type binding; Regulation of information transcription, DNA-regulation 2 dependent; Nucleus; homolog) 1 Apoptosis; Myogenesis; (S.cerevisiae) Chromatin silencing; Chromatin silencing complex; NM_003073SMARCB1 SWI/SNF Negative regulation of cell related, matrix cycle;Regulation of associated, actin transcription from Pol II dependentpromoter; Nuclear regulator of chromosome; chromatin, Nucleoplasm;Chromatin subfamily b, remodeling; DNA member 1 integration; NM_000345SNCA Synuclein, alpha Cytoplasm; Anti- (non A4 apoptosis; Centralnervous component of system development; amyloid precursor) NM_007315STAT1 Signal transducer Regulation of and activator of transcription,DNA- transcription 1, dependent; Nucleus; 91 kDa Cytoplasm;Intracellular signaling cascade; Regulation of cell cycle; Transcriptionfactor activity; Signal transducer activity; Transcription from Pol IIpromoter; Caspase activation; STAT protein nuclear translocation;Tyrosine phosphorylation of STAT protein; Hematopoietin/interferon-class (D200-domain) cytokine receptor signal transducer activity; I-kappaB kinase/NF-kappaB cascade; Response to pest, pathogen or parasite;NM_006354 TADA3L Transcriptional Nucleus; Regulation of cell adaptor 3(NGG1 cycle; Transcription factor homolog, yeast)- activity; Regulationof like transcription from Pol II promoter; NM_000594 TNF Tumor necrosisIntegral to membrane; factor (TNF Signal transduction; superfamily,Immune response; member 2) Regulation of transcrip- tion, DNA-dependent;Apoptosis; Anti-apoptosis; Inflammatory response; Response to virus;Soluble fraction; Cell-cell signaling; Tumor necrosis factor receptorbinding; Leukocyte cell adhesion; Necrosis; NM_003842 TNFRSF10B Tumornecrosis Integral to membrane; factor receptor Signal transduction;superfamily, Protein binding; Electron member 10b transporter activity;Induction of apoptosis; Regulation of apoptosis; Induction of apoptosisvia death domain receptors; Positive regulation of I- kappaBkinase/NF-kappaB cascade; Receptor activity; Iron ion binding; Electrontransport; TRAIL binding; Caspase activator activity; Caspaseactivation; Activation of NF-kappaB- inducing kinase; NM_000639 FASLGFas ligand (TNF Signal transduction; superfamily, Extracellular; Immunemember 6) response; Induction of apoptosis; Apoptosis; Positiveregulation of I- kappaB kinase/NF-kappaB cascade; Integral to plasmamembrane; Cell-cell signaling; Tumor necrosis factor receptor binding;NM_000546 TP53 Tumor protein ATP binding; Cell p53 proliferation;Regulation of (Li-Fraumeni transcription, DNA- syndrome) dependent;Protein binding; Negative regulation of cell cycle; Apoptosis;Mitochondrion; Transcription factor activity; Zinc ion binding; DNAdamage response, signal transduction resulting in induction ofapoptosis; Cell cycle arrest; Nucleolus; Cell cycle checkpoint; DNAstrand annealing activity; Copper ion binding; Nuclease activity; DNArecombination; Base- excision repair; Caspase activation via cytochromec; Cell aging; Cell differentiation; Induction of apoptosis by hormones;Negative regulation of cell growth; Nucleotide- excision repair;Regulation of mitochondrial membrane permeability; Proteintetramerization activity; Negative regulation of helicase activity;NM_005426 TP53BP2 Tumor protein Signal transduction; p53 Cytoplasm;Apoptosis; binding protein, Regulation of cell cycle; 2 SH3/SH2 adaptorprotein activity; NM_005427 TP73 Tumor protein Regulation of p73transcription, DNA- dependent; Nucleus; Protein binding; Negativeregulation of cell cycle; Apoptosis; Transcription factor activity; DNAdamage response, signal transduction resulting in induction ofapoptosis; Mismatch repair; NM_003722 TP73L Tumor protein Regulation ofp73-like transcription, DNA- dependent; Nucleus; Induction of apoptosis;Apoptosis; Transcription factor activity; Transcriptional activatoractivity; NM_021138 TRAF2 TNF receptor- Signal transduction; associatedfactor Protein complex assembly; 2 Apoptosis; Zinc ion binding; Signaltransducer activity; Ubiquitin-protein ligase activity; Proteinubiquitination; Ubiquitin ligase complex; NM_004295 TRAF4 TNF receptor-Nucleus; Apoptosis; Zinc associated factor ion binding; Ubiquitin- 4protein ligase activity; Protein ubiquitination; Ubiquitin ligasecomplex; Development; NM_004619 TRAF5 TNF receptor- Signal transduction;associated factor Apoptosis; Zinc ion 5 binding; Signal transduceractivity; Ubiquitin-protein ligase activity; Protein ubiquitination;Ubiquitin ligase complex; Positive regulation of I-kappaBkinase/NF-kappaB cascade; NM_000368 TSC1 Tuberous Cell adhesion; Rhoprotein sclerosis 1 signal transduction; Negative regulation of cellcycle; NM_000548 TSC2 Tuberous Plasma membrane; sclerosis 2 GTPaseactivator activity; Cell growth and/or maintenance; Negative regulationof cell cycle; Cytosol; Membrane fraction; Protein folding; Endocytosis;Unfolded protein binding; NM_000369 TSHR Thyroid Positive regulation ofcell stimulating proliferation; Signal hormone receptor transduceractivity; Integral to plasma membrane; Cell-cell signaling; G-proteinsignaling, coupled to cyclic nucleotide second messenger; HeterotrimericG-protein complex; Thyroid-stimulating hormone receptor activity;NM_000378 WT1 Wilms tumor 1 Nucleic acid binding; Regulation oftranscription, DNA- dependent; Nucleus; Negative regulation of cellcycle; Transcription factor activity; Zinc ion binding; NM_002046 GAPDHGlyceraldehyde- Cytoplasm; Oxidore- 3-phosphate ductase activity;dehydrogenase Glyceraldehyde-3- phosphate dehydrogenase(phosphorylating) activity; Glucose metabolism; Glycolysis; NM_004048B2M Beta-2- Extracellular; Immune microglobulin response; NM_007355HSP90AB1 Heat shock ATP binding; Protein protein 90 kDa binding;Cytoplasm; Heat alpha (cytosolic), shock protein activity; class Bmember Protein folding; TPR 1 domain binding; Nitric- oxide synthaseregulator activity; Positive regulation of nitric oxide biosynthesis;Unfolded protein binding; Response to unfolded protein; ATP binding;Protein binding; Cytoplasm; Heat shock protein activity; Proteinfolding; TPR domain binding; Nitric-oxide synthase regulator activity;Positive regulation of nitric oxide biosynthesis; Unfolded proteinbinding; Response to unfolded protein; NM_007355 HSP90AB1 Heat shock ATPbinding; Protein protein 90 kDa binding; Cytoplasm; Heat alpha(cytosolic), shock protein activity; class B member Protein folding; TPR1 domain binding; Nitric- oxide synthase regulator activity; Positiveregulation of nitric oxide biosynthesis; Unfolded protein binding;Response to unfolded protein; ATP binding; Protein binding; Cytoplasm;Heat shock protein activity; Protein folding; TPR domain binding;Nitric-oxide synthase regulator activity; Positive regulation of nitricoxide biosynthesis; Unfolded protein binding; Response to unfoldedprotein;

II. ASPECTS AND EMBODIMENTS OF THE INVENTION

In one aspect, therapeutic miR-34a, miR-34b, miR-34c, or miR-449, siRNAor shRNA compositions are provided that may be used to inhibit celldivision of a mammalian cell that has functional TP53 activity. Asdescribed in co-pending application PCT/US2008/62681 filed concurrentlyherewith, baseline levels of the one or more members of miR-34 arecorrelated with a TP53 pathway activity status when the obtained levelof miR-34 is related in a statistically significant fashion to thefunctional activity of TP53 or the functional activity of the TP53pathway.

A baseline level of miR-34 can be established by reference to a specificcell line wherein the cell line is known to have functional TP53activity or defective TP53 activity. Examples of cell lines havingfunctional TP53, include, but are not limited to, HCT116 (Vassilev etal., 2004, Science, 303:844-8), LOVO (Cottu et al., 1995, Cancer Res,13:2727-30), LS123 (Liu and Bodmer, 2006, PNAS, 103:976-81), RKO(Vassilev et al., 2004, Science, 303:844-8) and RKO-AS45-1 (Bamford, etal., 2004, Br. J. Cancer 91:355-58). Examples of cell lines havingdefective TP53 include, but are not limited to, HT29 (Rodrigues et al.,1990, PNAS, 87:7555-9), LS1034 (Liu and Bodmer, 2006, PNAS, 103:976-81),SW1417 (Liu and Bodmer, 2006, PNAS, 103:976-81), SW1116 (Liu and Bodmer,2006, PNAS, 103:976-81), and SW620 (Rodrigues et al., 1990, PNAS,87:7555-9). Alternatively, matched cell line pairs with and withoutfunctional TP53, such as those described in Example 1 herein, can betransfected with a nucleic acid vector encoding a shRNA hairpin moleculetargeting TP53 for gene silencing.

In other embodiments, multiple different cell samples can be pooledtogether and the resulting pool used to set the baseline level ofmiR-34, or alternatively, the baseline level can be obtained usingindividual miR-34 measurements from a plurality of different cellsamples using any of a variety of different statistical tests that areknown in the art. In still other embodiments, the baseline level ofmiR-34 is established based upon the level of one or more miR-34 membersmeasured in one or more cell or tissue samples of the subject or speciesof the subject.

In other embodiments, the p53 pathway status of a cell sample obtainedfrom a tumor sample is used to determine a course of treatment for apatient having cancer. For example, patients having tumors that areclassified as having a substantially active TP53 pathway status aretreated with a therapeutically sufficient amount of a compositioncomprising one or more DNA damaging agents. The one or more DNA damagingagents can comprise a topoisomerase I inhibitor, e.g., camptothecin; atopoisomerase II inhibitor, e.g., doxorubicin; a DNA binding agent,e.g., cisplatin; an anti-metabolite; or ionizing radiation.

In another embodiment, patients having tumors that are classified ashaving substantially inactive TP53 pathway status are treated with atherapeutically sufficient amount of a composition comprising one ormore DNA damaging agents in combination with an inhibitor of a proteinor gene capable of enhancing cell killing by the one or more DNAdamaging agents. Genes and proteins whose activity affects, eitherpositively or negatively, the sensitivity of TP53 pathway inactive cellsto DNA damaging agents are described in PCT Publication WO 2005/031002.

One embodiment of therapeutic treatment involves use of atherapeutically sufficient amount of a composition comprising a miR-34family member selected from miR-34a, miR-34b, miR-34c or miR-449 siRNAor shRNA to treat tumors classified as containing functional TP53. Suchtreatment may be in combination with one or more DNA damaging agents.

Therapeutic miR-34a, miR-34b, miR-34c, or miR-449, siRNA or shRNAcompositions comprise a guide strand contiguous nucleotide sequence ofat least 18 nucleotides, wherein said guide strand comprises a seedregion consisting of nucleotide positions 1 to 12, wherein position 1represents the 5′ end of said guide strand and wherein said seed regioncomprises a nucleotide sequence of at least six contiguous nucleotidesthat is identical to six contiguous nucleotides within a sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:9 and SEQ ID NO:31.

In some embodiments, therapeutic miR-34a, miR-34b, miR-34c, or miR-449,siRNA or shRNA compositions comprise a guide a guide strand nucleotidesequence of 18 to 25 nucleotides, said guide strand nucleotide sequencecomprising a seed region nucleotide sequence and a non-seed regionnucleotide sequence, said seed region consisting essentially ofnucleotide positions 1 to 12 and said non-seed region consistingessential of nucleotide positions 13 to the 3′ end of said guide strand,wherein position 1 of said guide strand represents the 5′ end of saidguide strand, wherein said seed region further comprises a consecutivenucleotide sequence of at least 6 nucleotides that is identical insequence to a nucleotide sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:31 and wherein saidisolated nucleic acid molecule has at least one nucleotide sequencedifference compared to a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:29.

In some embodiments, therapeutic miR-34a, miR-34b, miR-34c, or miR-449,siRNA or shRNA compositions comprise synthetic duplex microRNA mimeticscomprising: (i) a guide strand nucleic acid molecule consisting of anucleotide sequence of 18 to 25 nucleotides, said guide strandnucleotide sequence comprising a seed region nucleotide sequence and anon-seed region nucleotide sequence, said seed region consisting ofnucleotide positions 1 to 12 and said non-seed region consisting ofnucleotide positions 13 to the 3′ end of said guide strand, whereinposition 1 of said guide strand represents the 5′ end of said guidestrand, wherein said seed region further comprises a consecutivenucleotide sequence of at least 6 nucleotides that is identical to aseed region sequence of a naturally occurring microRNA; and (ii) apassenger strand nucleic acid molecule consisting of a nucleotidesequence of 18 to 25 nucleotides, said passenger strand comprising anucleotide sequence that is essentially complementary to the guidestrand, wherein said passenger strand nucleic acid molecule has onenucleotide sequence difference compared with the true reverse complementsequence of the seed region of the guide strand, wherein the onenucleotide difference is located within nucleotide position 13 to the 3′end of the passenger strand.

In certain embodiments, at least one of the two strands furthercomprises a 1-4, preferably a 2 nucleotide, 3′ overhang. The nucleotideoverhang can include any combination of a thymine, uracil, adenine,guanine, or cytosine, or derivatives or analogues thereof. Thenucleotide overhang in certain aspects is a 2 nucleotide overhang, whereboth nucleotides are thymine. Importantly, when the dsRNA comprising thesense and antisense strands is administered, it directs target specificinterference and bypasses an interferon response pathway.

In order to enhance the stability of the short interfering nucleicacids, the 3′ overhangs can also be stabilized against degradation. Inone embodiment, the 3′ overhangs are stabilized by including purinenucleotides, such as adenosine or guanosine nucleotides. Alternatively,substitution of pyrimidine nucleotides by modified analogues, e.g.,substitution of uridine nucleotides in the 3′ overhangs with2′-deoxythymidine, is tolerated and does not affect the efficiency ofRNAi degradation. In particular, the absence of a 2′ hydroxyl in the2′-deoxythymidine significantly enhances the nuclease resistance of the3′ overhang in tissue culture medium.

As used herein, a “3′ overhang” refers to at least one unpairednucleotide extending from the 3′ end of an siRNA sequence. The 3′overhang can include ribonucleotides or deoxyribonucleotides or modifiedribonucleotides or modified deoxyribonucleotides. The 3′ overhang ispreferably from 1 to about 5 nucleotides in length, more preferably from1 to about 4 nucleotides in length and most preferably from about 2 toabout 4 nucleotides in length. The 3′ overhang can occur on the sense orantisense sequence, or on both sequences, of an RNAi construct. Thelength of the overhangs can be the same or different for each strand ofthe duplex. Most preferably, a 3′ overhang is present on both strands ofthe duplex, and the overhang for each strand is 2 nucleotides in length.For example, each strand of the duplex can comprise 3′ overhangs ofdithymidylic acid (“tt”) or diuridylic acid (“uu”).

Another aspect of the invention provides chemically modified siRNAconstructs. For example, the siRNA agent can include a non-nucleotidemoiety. A chemical modification or other non-nucleotide moiety canstabilize the sense (guide strand) and antisense (passenger strand)sequences against nucleolytic degradation. Additionally, conjugates canbe used to increase uptake and target uptake of the siRNA agent toparticular cell types. Thus, in one embodiment, the siRNA agent includesa duplex molecule wherein one or more sequences of the duplex moleculeis chemically modified. Non-limiting examples of such chemicalmodifications include phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal base” nucleotides, “acyclic” nucleotides,5′-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxyabasic residue incorporation. These chemical modifications, when used insiRNA agents, can help to preserve RNAi activity of the agents in cellsand can increase the serum stability of the siRNA agents.

In one embodiment, the first, and optionally or preferably the firsttwo, internucleotide linkages at the 5′ end of the antisense and/orsense sequences are modified, preferably by a phosphorothioate. Inanother embodiment, the first, and perhaps the first two, three, orfour, internucleotide linkages at the 3′ end of a sense and/or antisensesequence are modified, for example, by a phosphorothioate. In anotherembodiment, the 5′ end of both the sense and antisense sequences, andthe 3′ end of both the sense and antisense sequences are modified asdescribed.

In some embodiments of the invention, TP53 pathway status relates todetermining degree to which the TP53 pathway is active or inactivewithin a cell or population of cells. For example, one measure ofwhether a cell has an active TP53 pathway is that activation of TP53 byultraviolet or ionizing radiation, or other DNA-damaging agents, such aschemotherapeutic agents, results in some degree of cell cycle arrestand/or apoptosis. Cells having an impaired or inactive TP53 pathwaystatus are unable to arrest cell division or initiate apoptosisfollowing cellular stress compared to cells having a functional oractive TP53 pathway. TP53 pathway status may also be characterized bymeasuring a defect or change in expression of one or more genes orproteins that are members of the TP53 pathway, such as those set forthin Table 1 above. In some embodiments of the invention, TP53 pathwaystatus may be classified into two status categories, such as, forexample, substantially functional (i.e., able to elicit TP53-mediatedcell cycle arrest in the presence of genotoxic stress or able toactivate a TP53-responsive reporter system (e.g., p53RE-bla; Catalog No.K1193 (Invitrogen Corporation, Carlsbad, Calif.)) and substantiallynonfunctional (e.g., unable to elicit TP53-mediated cell cycle arrest inthe presence of genotoxic stress or unable to activate a TP53-responsivereporter system), based upon measurement of one or more miR-34 levels ina cell sample.

Alternatively, TP53 functional status may be classified into three ormore functional categories, such as for example, high TP53 pathwayactivity, medium TP53 pathway activity, or low TP53 pathway activity,based upon the level of miR-34 measured in a cell. Threshold levels foreach such TP53 pathway status category can be set by measuring orobtaining a range of miR-34 levels from a plurality of different celltypes or cell samples whose TP53 pathway function has been determined orevaluated based on functional biological measurement of TP53 pathwayfunction.

In one embodiment of this aspect of the invention, the miR-34 moleculelevel that is measured or obtained is selected from the group consistingof miR-34a (SEQ ID NO:1), miR-34b (SEQ ID NO:4), miR-34c (SEQ ID NO:7),and precursor RNAs thereof (SEQ ID NO:2; SEQ ID NO:5 and SEQ ID NO:8,respectively).

Another aspect of the invention provides a method of inhibiting celldivision of a mammalian cell comprising introducing into said cell aneffective amount of a small interfering nucleic acid (siNA), whereinsaid siNA comprises a guide strand contiguous nucleotide sequence of atleast 18 nucleotides, wherein said guide strand comprises a seed regionconsisting of nucleotide positions 1 to 12, wherein position 1represents the 5′ end of said guide strand and wherein said seed regioncomprises a nucleotide sequence of at least six contiguous nucleotidesthat is identical to six contiguous nucleotides within a sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:9 and SEQ ID NO:31.

In one embodiment, the siNA is a duplex RNA molecule that is introducedinto said cell by transfection. In some embodiments, the introduced siNAincludes one or more chemically modified nucleotides. An effectiveamount of siNA is the amount sufficient to cause a measurable change inthe detected level of one or more gene transcripts that are regulated byone or more members of the miR-34 family. In one embodiment, the genetranscripts regulated by one or more members of the miR-34 family areselected from Table 5.

In another embodiment, cell division is inhibited by introduction of anucleic acid vector molecule expressing an shRNA gene, wherein the shRNAtranscription product acts as an RNAi agent. The shRNA gene may encode amicroRNA precursor RNA, such as, for example, SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:8, or SEQ ID NO:30. Alternatively, the shRNA gene may encodeany other RNA sequence that is susceptible to processing by endogenouscellular RNA processing enzymes into an active siRNA sequence, whereinthe seed region of the active siRNA sequence contains at least a sixcontiguous nucleotide sequence that is identical to a six contiguousnucleotide sequence within SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQID NO:31. Examples of vectors and transcription promoter sequencesuseful for expression of shRNA genes are well known in the art(Paddison, et al., 2004, Nature 4: 28-31; Silva et al., 2005, Nat.Genet. 37:1281-88; Bernards et al., 2006, Nat. Methods 3:701-06). Aneffective amount of shRNA is the amount sufficient to cause a measurablechange in the detected level of one or more gene transcripts that areregulated by one or more members of the miR-34 family. In oneembodiment, the gene transcripts regulated by one or more members of themiR-34 family are selected from Table 5.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising, or consisting essentially of, a guide strandnucleotide sequence of 18 to 25 nucleotides, said guide strandnucleotide sequence comprising a seed region nucleotide sequence and anon-seed region nucleotide sequence, said seed region consistingessentially of nucleotide positions 1 to 12 and said non-seed regionconsisting essentially of nucleotide positions 13 to the 3′ end of saidguide strand, wherein position 1 of said guide strand represents the 5′end of said guide strand, wherein said seed region further comprises aconsecutive nucleotide sequence of at least 6 nucleotides that isidentical in sequence to a nucleotide sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:31and wherein said isolated nucleic acid molecule has at least onenucleotide sequence difference, compared to a nucleotide sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQID NO:3.

In one embodiment, the isolated nucleic acid molecule consistsessentially of a guide strand nucleotide sequence of 19 to 23nucleotides, said guide strand nucleotide sequence comprising a seedregion nucleotide sequence and a non-seed region nucleotide sequence,said seed region consisting essentially of nucleotide positions 1 to 10and said non-seed region consisting essentially of nucleotide positions11 to the 3′ end of said guide strand, wherein position 1 of said guidestrand represents the 5′ end of said guide strand, wherein said seedregion further comprises a consecutive nucleotide sequence of at least 6nucleotides that is identical in sequence to a nucleotide sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:9, and SEQ ID NO:31, and wherein said isolated nucleic acid moleculehas at least one nucleotide sequence difference, compared to anucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, and SEQ ID NO:3.

In another aspect, the invention provides isolated synthetic duplexmicroRNA mimetics and methods of making synthetic duplex microRNAmimetics. As described herein, it has been demonstrated that a syntheticduplex microRNA mimetic comprising a guide strand with the sequencecorresponding to natural mature miR34a (SEQ ID NO:1), and a syntheticpassenger strand (SEQ ID NO:12) that is essentially complementary to themiR34a natural mature guide strand, except for a single base mismatchlocated in the 3′ end of the sequence (assymetric passenger strand) wasmore effective at inducing a cell cycle phenotype when transfected intocells, than a duplex consisting of the natural miR-34a guide strand (SEQID NO:1) and the natural miR-34a passenger strand (SEQ ID NO:35), asdemonstrated in Example 5. While not wishing to be bound by theory, itis believed that the presence of a mismatch in the passenger strand mayfacilitate entry into RISC.

In accordance with the foregoing, in one embodiment, the inventionprovides an isolated synthetic duplex microRNA mimetic comprising (i) aguide strand nucleic acid molecule consisting of a nucleotide sequenceof 18 to 25 nucleotides, said guide strand nucleotide sequencecomprising a seed region nucleotide sequence and a non-seed regionnucleotide sequence, said seed region consisting of nucleotide positions1 to 12 and said non-seed region consisting of nucleotide positions 13to the 3′ end of said guide strand, wherein position 1 of said guidestrand represents the 5′ end of said guide strand, wherein said seedregion further comprises a consecutive nucleotide sequence of at least 6nucleotides that is identical to a seed region sequence of a naturallyoccurring microRNA; and (ii) a passenger strand nucleic acid moleculeconsisting of a nucleotide sequence of 18 to 25 nucleotides, saidpassenger strand comprising a nucleotide sequence that is essentiallycomplementary to the guide strand, wherein said passenger strand nucleicacid molecule has one nucleotide sequence difference compared with thetrue reverse complement sequence of the seed region of the guide strand,wherein the one nucleotide difference is located within nucleotideposition 13 to the 3′ end of the passenger strand.

In accordance with this aspect of the invention, a synthetic duplexmimetic may be generated for any naturally occurring microRNA.Computational and molecular cloning approaches have revealed hundreds ofmicroRNAs that are expressed at various levels in a variety oforganisms. Over 200 different mammalian microRNAs have been identified,as described in the “miRBase sequence database” which is publiclyaccessible on the World Wide Web at the Wellcome Trust Sanger Institutewebsite at http://microrna.sanger.ac.uk/sequences/. A list of exemplarymicroRNA species is also described in the following references: Ambroset al., RNA 9: 277-279 (2003); Griffith-Jones, Nucleic Acid Res. 32:D109-D111 (2004); Griffith-Jones, Nucleic Acids Res. 34:D140-D144(2006); Lagos-Quintana et al., Curr Biol. 12(9): 735-9 (2002); Lim. L.P. et al., Science 299 (5612): 1540 (2003). The synthetic duplexmicroRNA mimetics of this aspect of the invention may be used tomodulate the level of microRNA responsive target sites for any givenmicroRNA. The synthetic duplex microRNA mimetics of this aspect of theinvention may be included in compositions with a delivery agent, such aslipid nanoparticles, as described herein.

In one embodiment of this aspect of the invention, the guide strandcomprises a seed region comprising a consecutive nucleotide sequence ofat least 6 nucleotides that is identical in sequence to a nucleotidesequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO:9 and SEQ ID NO:31. In one embodiment, the guide strandsequence is selected from the group consisting of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:7 and SEQ ID NO:29.

In accordance with this aspect of the invention, the passenger strand isa nucleic acid molecule consisting of a nucleotide sequence of 18 to 25nucleotides. The nucleotide sequence of the passenger strand isessentially complementary to the guide strand, wherein the passengerstrand has one nucleotide sequence difference as compared with the truereverse complement sequence of the seed region of the guide strand. Asused herein, the term “essentially complementary” with reference toguide strand refers to a passenger strand that is the reverse complementof a guide strand with a one base mismatch (one nucleotide sequencedifference) with the true reverse complement of the guide strand seedsequence (positions 1 to 12 of the guide strand), which is located atthe 3′ end of the passenger strand (from position 13 to the 3′ end). Insome embodiments, the one nucleotide sequence difference is locatedwithin 6 nucleotides of the 3′ end of the passenger strand. In oneembodiment, the one nucleotide sequence difference is located 6nucleotides from the 3′ end of the passenger strand. In one embodiment,the one nucleotide sequence difference is located 5 nucleotides from the3′ end of the passenger strand. In one embodiment, the one nucleotidesequence difference is located 4 nucleotides from the 3′ end of thepassenger strand. In one embodiment, the one nucleotide sequencedifference is located 3 nucleotides from the 3′ end of the passengerstrand. In one embodiment, the one nucleotide sequence difference islocated 2 nucleotides from the 3′ end of the passenger strand.

In some embodiments, the nucleotide sequence of the passenger strand isessentially complementary to the reverse complement of the sequence ofthe guide strand, wherein the 5′ end of the passenger strand iscomplementary to a position 1 to 4 bases from the 3′ end of the guidestrand, thereby forming a 3′ overhang on one end of the duplex when theguide strand and passenger strand are annealed together.

In some embodiments, the nucleotide sequence is essentiallycomplementary to the reverse complement of the sequence of the guidestrand, wherein the 3′ end of the passenger strand extends from 1 to 4bases beyond the 5′ end of the guide strand, thereby forming a 3′overhang on one end of the duplex when the guide strand and passengerstrand are annealed together.

In one embodiment, the isolated synthetic duplex comprises guide strandSEQ ID NO:1 and passenger strand SEQ ID NO:12. In one embodiment, theisolated synthetic duplex comprises guide strand SEQ ID NO:4 andpassenger strand SEQ ID NO:17. In one embodiment, the isolated syntheticduplex comprises guide strand SEQ ID NO:7 and passenger strand SEQ IDNO:22. In one embodiment, the isolated synthetic duplex comprises guidestrand SEQ ID NO:29 and passenger strand SEQ ID NO:32.

In another aspect, the invention provides methods of making a syntheticduplex microRNA mimetic. The methods according to this aspect of theinvention comprise annealing an isolated guide strand nucleic acidmolecule with an isolated passenger strand nucleic acid molecule to forma synthetic duplex microRNA mimetic, wherein (i) the isolated guidestrand nucleic acid molecule consists of a nucleotide sequence of 18 to25 nucleotides, said guide strand nucleotide sequence comprising a seedregion nucleotide sequence and a non-seed region nucleotide sequence,said seed region consisting of nucleotide positions 1 to 12 and saidnon-seed region consisting of nucleotide positions 13 to the 3′ end ofsaid guide strand, wherein position 1 of said guide strand representsthe 5′ end of said guide strand, wherein said seed region furthercomprises a consecutive nucleotide sequence of at least 6 nucleotidesthat is identical to a seed region sequence of a naturally occurringmicroRNA; and (ii) the isolated passenger strand nucleic acid moleculeconsists of a nucleotide sequence of 18 to 25 nucleotides, saidpassenger strand comprising a nucleotide sequence that is essentiallycomplementary to the guide strand, wherein said passenger strand nucleicacid molecule has one nucleotide sequence difference compared with thetrue reverse complement sequence of the seed region of the guide strand,wherein the one nucleotide difference is located within nucleotideposition 13 to the 3′ end of the passenger strand.

III. NUCLEIC ACID MOLECULES

As used herein a “nucleobase” refers to a heterocyclic base, such as,for example, a naturally occurring nucleobase (i.e., an A, T, G, C, orU) found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in a manner that may substitute for a naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, a purine or pyrimidinesubstituted by one or more of an alkyl, carboxyalkyl, amino, hydroxyl,halogen (i.e., fluoro, chloro, bromo, or iodo), thiol or alkylthiolmoeity. Preferred alkyl (e.g., alkyl, carboxyalkyl, etc.) moietiescomprise of from about 1, about 2, about 3, about 4, about 5, to about 6carbon atoms. Other non-limiting examples of a purine or pyrimidineinclude a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil, axanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenine, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like. A nucleobasemay be comprised in a nucleoside or nucleotide, using any chemical ornatural synthesis method described herein or known to one of ordinaryskill in the art. Such nucleobase may be labeled or it may be part of amolecule that is labeled and contains the nucleobase.

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), including,but not limited to, a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992, “DNA replication,” Freeman and Company, New York, N.Y.).

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety.” A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. Other types of attachments are known in the art, particularlywhen a nucleotide comprises derivatives or analogs of a naturallyoccurring 5-carbon sugar or phosphorus moiety.

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. Asused herein a “derivative” refers to a chemically modified or alteredform of a naturally occurring molecule, while the terms “mimic” or“analog” refer to a molecule that may or may not structurally resemble anaturally occurring molecule or moiety, but possesses similar functions.As used herein, a “moiety” generally refers to a smaller chemical ormolecular component of a larger chemical or molecular structure.Nucleobase, nucleoside and nucleotide analogs or derivatives are wellknown in the art, and have been described (see for example, Scheit,1980, “Nucleotide Analogs: Synthesis and Biological Function,” Wiley,N.Y.).

Additional non-limiting examples of nucleosides, nucleotides, or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in: U.S. Pat. No. 5,681,947, which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167, which describe nucleic acids incorporating fluorescentanalogs of nucleosides found in DNA or RNA, particularly for use asfluorescent nucleic acid probes; U.S. Pat. No. 5,614,617, whichdescribes oligonucleotide analogs with substitutions on pyrimidine ringsthat possess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663,5,872,232 and 5,859,221, which describe oligonucleotide analogs withmodified 5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties)used in nucleic acid detection; U.S. Pat. No. 5,446,137, which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165, whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606, which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697, which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847, which describe the linkage of asubstituent moeity, which may comprise a drug or label, to the 2′ carbonof an oligonucleotide to provide enhanced nuclease stability and abilityto deliver drugs or detection moieties; U.S. Pat. No. 5,223,618, whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of an adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967, which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobes; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240, which describe oligonucleotides with a three or four atomlinker moiety replacing phosphodiester backbone moiety used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988, which describes a hydrophobic carrier agent attachedto the 2′-O position of oligonucleotides to enhance their membranepermeability and stability; U.S. Pat. No. 5,214,136, which describesoligonucleotides conjugated to anthraquinone at the 5′ terminus thatpossesses enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922, which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H; and U.S. Pat. No. 5,708,154, which describes RNAlinked to a DNA to form a DNA-RNA hybrid; and U.S. Pat. No. 5,728,525,which describes the labeling of nucleoside analogs with a universalfluorescent label.

Additional teachings for nucleoside analogs and nucleic acid analogs areU.S. Pat. No. 5,728,525, which describes nucleoside analogs that areend-labeled; and U.S. Pat. Nos. 5,637,683, 6,251,666 (L-nucleotidesubstitutions), and 5,480,980 (7-deaza-2′ deoxyguanosine nucleotides andnucleic acid analogs thereof).

shRNA Mediated Suppression

Alternatively, certain of the nucleic acid molecules of the instantinvention can be expressed within cells from eukaryotic promoters (e.g.,Izant and Weintraub, 1985, Science, 229:345; McGarry and Lindquist,1986, Proc. Natl. Acad. Sci., USA 83:399; Scanlon et al., 1991, Proc.Natl. Acad. Sci. USA, 88:10591-95; Kashani-Sabet et al., 1992, AntisenseRes. Dev., 2:3-15; Dropulic et al., 1992, J. Virol., 66:1432-41;Weerasinghe et al., 1991, J. Virol., 65:5531-4; Ojwang et al., 1992,Proc. Natl. Acad. Sci. USA, 89:10802-06; Chen et al., 1992, NucleicAcids Res., 20:4581 89; Sarver et al., 1990 Science, 247:1222-25;Thompson et al., 1995, Nucleic Acids Res., 23:2259; Good et al., 1997,Gene Therapy, 4:45). Those skilled in the art realize that any nucleicacid can be expressed in eukaryotic cells from the appropriate DNA/RNAvector. The activity of such nucleic acids can be augmented by theirrelease from the primary transcript by an enzymatic nucleic acid (Draperet al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa etal., 1992, Nucleic Acids Symp. Ser., 27:15-6; Taira et al., 1991,Nucleic Acids Res., 19:5125-30; Ventura et al., 1993, Nucleic AcidsRes., 21:3249-55; Chowrira et al., 1994, J. Biol. Chem., 269:25856).Gene therapy approaches specific to the CNS are described by Blesch etal., 2000, Drug News Perspect., 13:269-280; Peterson et al., 2000, Cent.Nerv. Syst. Dis., 485:508; Peel and Klein, 2000, J. Neurosci. Methods,98:95-104; Hagihara et al., 2000, Gene Ther., 7:759-763; and Herrlingeret al., 2000, Methods Mol. Med., 35:287-312. AAV-mediated delivery ofnucleic acid to cells of the nervous system is further described byKaplitt et al., U.S. Pat. No. 6,180,613.

In another aspect of the invention, RNA molecules of the presentinvention are preferably expressed from transcription units (see forexample Couture et al., 1996, TIG., 12:510) inserted into DNA or RNAvectors. The recombinant vectors are preferably DNA plasmids or viralvectors. Ribozyme expressing viral vectors can be constructed based on,but not limited to, adeno-associated virus, retrovirus, adenovirus, oralphavirus. Preferably, the recombinant vectors capable of expressingthe nucleic acid molecules are delivered as described above, and persistin target cells. Alternatively, viral vectors can be used that providefor transient expression of nucleic acid molecules. Such vectors can berepeatedly administered as necessary. Once expressed, the nucleic acidmolecule binds to the target mRNA. Delivery of nucleic acid moleculeexpressing vectors can be systemic, such as by intravenous orintramuscular administration, by administration to target cellsex-planted from the patient or subject followed by reintroduction intothe patient or subject, or by any other means that would allow forintroduction into the desired target cell (for a review see Couture etal., 1996, TIG., 12:510).

In one aspect, the invention features an expression vector comprising anucleic acid sequence encoding at least one of the nucleic acidmolecules of the instant invention. The nucleic acid sequence encodingthe nucleic acid molecule of the instant invention is operably linked ina manner which allows expression of that nucleic acid molecule.

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II, or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II, or III termination region); c) a nucleicacid sequence encoding at least one of the nucleic acid molecules of theinstant invention; and wherein said sequence is operably linked to saidinitiation region and said termination region, in a manner which allowsexpression and/or delivery of said nucleic acid molecule. The vector canoptionally include an open reading frame (ORF) for a protein operablylinked on the 5′ side or the 3′-side of the sequence encoding thenucleic acid molecule of the invention; and/or an intron (interveningsequences).

Transcription of the nucleic acid molecule sequences are driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87:6743-7; Gaoand Huang, 1993, Nucleic Acids Res., 21:2867-72; Lieber et al., 1993,Methods Enzymol., 217:47-66; Zhou et al., 1990, Mol. Cell. Biol.,10:4529-37).

Several investigators have demonstrated that nucleic acid moleculesencoding shRNAs or microRNAs expressed from such promoters can functionin mammalian cells (Brummelkamp et al., 2002, Science 296:550-553;Paddison et al., 2004, Nat. Methods 1:163-67; McIntyre and Fanning 2006BMC Biotechnology (January 5) 6:1; Taxman et al., 2006 BMC Biotechnology(January 24) 6:7). The above shRNA or microRNA transcription units canbe incorporated into a variety of vectors for introduction intomammalian cells, including but not restricted to, plasmid DNA vectors,viral DNA vectors (such as adenovirus or adeno-associated virusvectors), or viral RNA vectors (such as retroviral or alphavirusvectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisingnucleic acid sequence encoding at least one of the nucleic acidmolecules of the invention, in a manner which allows expression of thatnucleic acid molecule. The expression vector comprises in oneembodiment: a) a transcription initiation region; b) a transcriptiontermination region; c) a nucleic acid sequence encoding at least onesaid nucleic acid molecule; and wherein said sequence is operably linkedto said initiation region and said termination region, in a manner whichallows expression and/or delivery of said nucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; d) a nucleic acid sequence encoding at leastone said nucleic acid molecule, wherein said sequence is operably linkedto the 3′-end of said open reading frame; and wherein said sequence isoperably linked to said initiation region, said open reading frame, andsaid termination region, in a manner which allows expression and/ordelivery of said nucleic acid molecule. In yet another embodiment, theexpression vector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; d) a nucleic acidsequence encoding at least one said nucleic acid molecule; and whereinsaid sequence is operably linked to said initiation region, said intronand said termination region, in a manner which allows expression and/ordelivery of said nucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; e) a nucleic acid sequenceencoding at least one said nucleic acid molecule, wherein said sequenceis operably linked to the 3′-end of said open reading frame; and whereinsaid sequence is operably linked to said initiation region, said intron,said open reading frame, and said termination region, in a manner whichallows expression and/or delivery of said nucleic acid molecule.

IV. MODIFIED SINA MOLECULES

Any of the siNA constructs described herein can be evaluated andmodified as described below.

An siNA construct may be susceptible to cleavage by an endonuclease orexonuclease, such as, for example, when the siNA construct is introducedinto the body of a subject. Methods can be used to determine sites ofcleavage, e.g., endo- and exonucleolytic cleavage on an RNAi constructand to determine the mechanism of cleavage. An siNA construct can bemodified to inhibit such cleavage.

Exemplary modifications include modifications that inhibitendonucleolytic degradation, including the modifications describedherein. Particularly favored modifications include: 2′ modification,e.g., a 2′-O-methylated nucleotide or 2′-deoxy nucleotide (e.g., 2′deoxy-cytodine), or a 2′-fluoro, difluorotoluoyl, 5-Me-2′-pyrimidines,5-allyamino-pyrimidines, 2′-O-methoxyethyl, 2′-hydroxy, or 2′-ara-fluoronucleotide, or a locked nucleic acid (LNA), extended nucleic acid (ENA),hexose nucleic acid (HNA), or cyclohexene nucleic acid (CeNA). In oneembodiment, the 2′ modification is on the uridine of at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide, at least one5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, or at least one5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, or on the cytidine of atleast one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, at least one5′-cytidine-cytidine-3′ (5′-CC-3′) dinucleotide, or at least one5′-cytidine-uridine-3′ (5′-CU-3′) dinucleotide. The 2′ modification canalso be applied to all the pyrimidines in an siNA construct. In onepreferred embodiment, the 2′ modification is a 2′OMe modification on thesense strand of an siNA construct. In a more preferred embodiment, the2′ modification is a 2′ fluoro modification, and the 2′ fluoro is on thesense (passenger) or antisense (guide) strand or on both strands.

Modification of the backbone, e.g., with the replacement of an O with anS, in the phosphate backbone, e.g., the provision of a phosphorothioatemodification can be used to inhibit endonuclease activity. In someembodiments, an siNA construct has been modified by replacing one ormore ribonucleotides with deoxyribonucleotides. Preferably, adjacentdeoxyribonucleotides are joined by phosphorothioate linkages, and thesiNA construct does not include more than four consecutivedeoxyribonucleotides on the sense or the antisense strands. Replacementof the U with a C5 amino linker; replacement of an A with a G (sequencechanges are preferred to be located on the sense strand and not theantisense strand); or modification of the sugar at the 2′, 6′, 7′, or 8′position can also inhibit endonuclease cleavage of the siNA construct.Preferred embodiments are those in which one or more of thesemodifications are present on the sense but not the antisense strand, orembodiments where the antisense strand has fewer of such modifications.

Exemplary modifications also include those that inhibit degradation byexonucleases. In one embodiment, an siNA construct includes aphosphorothioate linkage or P-alkyl modification in the linkages betweenone or more of the terminal nucleotides of an siNA construct. In anotherembodiment, one or more terminal nucleotides of an siNA constructinclude a sugar modification, e.g., a 2′ or 3′ sugar modification.Exemplary sugar modifications include, for example, a 2′-O-methylatednucleotide, 2′-deoxy nucleotide (e.g., deoxy-cytodine),2′-deoxy-2′-fluoro (2′-F) nucleotide, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O—N-methylacetamido (2′-O—NMA),2′-O-dimethylaminoethlyoxyethyl (2′-DMAEOE), 2′-β-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-AP), 2′-hydroxy nucleotide,or a 2′-ara-fluoro nucleotide, or a locked nucleic acid (LNA), extendednucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleicacid (CeNA). A 2′ modification is preferably 2′OMe, more preferably, 2′fluoro.

The modifications described to inhibit exonucleolytic cleavage can becombined onto a single siNA construct. For example, in one embodiment,at least one terminal nucleotide of an siNA construct has aphosphorothioate linkage and a 2′ sugar modification, e.g., a 2′F or2′OMe modification. In another embodiment, at least one terminalnucleotide of an siNA construct has a 5′ Me-pyrimidine and a 2′ sugarmodification, e.g., a 2′F or 2′OMe modification.

To inhibit exonuclease cleavage, an siNA construct can include anucleobase modification, such as a cationic modification, such as a3′-abasic cationic modification. The cationic modification can be, e.g.,an alkylamino-dT (e.g., a C6 amino-dT), an allylamino conjugate, apyrrolidine conjugate, a pthalamido or a hydroxyprolinol conjugate, onone or more of the terminal nucleotides of the siNA construct. In oneembodiment, an alkylamino-dT conjugate is attached to the 3′ end of thesense or antisense strand of an RNAi construct. In another embodiment, apyrrolidine linker is attached to the 3′ or 5′ end of the sense strand,or the 3′ end of the antisense strand. In one embodiment, an allyl amineuridine is on the 3′ or 5′ end of the sense strand, and not on the 5′end of the antisense strand.

In one embodiment, the siNA construct includes a conjugate on one ormore of the terminal nucleotides of the siNA construct. The conjugatecan be, for example, a lipophile, a terpene, a protein binding agent, avitamin, a carbohydrate, a retinoid, or a peptide. For example, theconjugate can be naproxen, nitroindole (or another conjugate thatcontributes to stacking interactions), folate, ibuprofen, cholesterol,retinoids, PEG, or a C5 pyrimidine linker. In other embodiments, theconjugates are glyceride lipid conjugates (e.g., a dialkyl glyceridederivative), vitamin E conjugates, or thio-cholesterols. In oneembodiment, conjugates are on the 3′ end of the antisense strand, or onthe 5′ or 3′ end of the sense strand and the conjugates are not on the3′ end of the antisense strand and on the 3′ end of the sense strand.

In one embodiment, the conjugate is naproxen, and the conjugate is onthe 5′ or 3′ end of the sense or antisense strands. In one embodiment,the conjugate is cholesterol, and the conjugate is on the 5′ or 3′ endof the sense strand and not present on the antisense strand. In someembodiments, the cholesterol is conjugated to the siNA construct by apyrrolidine linker, or serinol linker, aminooxy, or hydroxyprolinollinker. In other embodiments, the conjugate is a dU-cholesterol, orcholesterol is conjugated to the siNA construct by a disulfide linkage.In another embodiment, the conjugate is cholanic acid, and the cholanicacid is attached to the 5′ or 3′ end of the sense strand, or the 3′ endof the antisense strand. In one embodiment, the cholanic acid isattached to the 3′ end of the sense strand and the 3′ end of theantisense strand. In another embodiment, the conjugate is PEG5, PEG20,naproxen or retinol.

In another embodiment, one or more terminal nucleotides have a 2′-5′linkage. In certain embodiments, a 2′-5′ linkage occurs on the sensestrand, e.g., the 5′ end of the sense strand.

In one embodiment, an siNA construct includes an L-sugar, preferably atthe 5′ or 3′ end of the sense strand.

In one embodiment, an siNA construct includes a methylphosphonate at oneor more terminal nucleotides to enhance exonuclease resistance, e.g., atthe 3′ end of the sense or antisense strands of the construct.

In one embodiment, an siRNA construct has been modified by replacing oneor more ribonucleotides with deoxyribonucleotides. In anotherembodiment, adjacent deoxyribonucleotides are joined by phosphorothioatelinkages. In one embodiment, the siNA construct does not include morethan four consecutive deoxyribonucleotides on the sense or the antisensestrands. In another embodiment, all of the ribonucleotides have beenreplaced with modified nucleotides that are not ribonucleotides.

In some embodiments, an siNA construct having increased stability incells and biological samples includes a difluorotoluoyl (DFT)modification, e.g., 2,4-difluorotoluoyl uracil, or a guanidine toinosine substitution.

The methods can be used to evaluate a candidate siNA, e.g., a candidatesiRNA construct, which is unmodified or which includes a modification,e.g., a modification that inhibits degradation, targets the dsRNAmolecule, or modulates hybridization. Such modifications are describedherein. A cleavage assay can be combined with an assay to determine theability of a modified or non-modified candidate to silence the targettranscript. For example, one might (optionally) test a candidate toevaluate its ability to silence a target (or off-target sequence),evaluate its susceptibility to cleavage, modify it (e.g., as describedherein, e.g., to inhibit degradation) to produce a modified candidate,and test the modified candidate for one or both of the ability tosilence and the ability to resist degradation. The procedure can berepeated. Modifications can be introduced one at a time or in groups. Itwill often be convenient to use a cell-based method to monitor theability to silence a target RNA. This can be followed by a differentmethod, e.g., a whole animal method, to confirm activity.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990,Nature 344:565; Pieken et al., 1991, Science 253:314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17:334; Burgin et al., 1996,Biochemistry, 35:14090; Usman et al., International Publication No. WO93/15187; and Rossi et al., International Publication No. WO 91/03162;Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074;and Vargeese et al., US 2006/021733). All of the above referencesdescribe various chemical modifications that can be made to the base,phosphate and/or sugar moieties of the nucleic acid molecules describedherein. Modifications that enhance their efficacy in cells, and removalof bases from nucleic acid molecules to shorten oligonucleotidesynthesis times and reduce chemical requirements are desired.

Chemically modified siNA molecules for use in modulating or attenuatingexpression of two or more genes down-regulated by one or more miR-34family member are also within the scope of the invention. Describedherein are isolated siNA agents, e.g., RNA molecules (chemicallymodified or not, double-stranded, or single-stranded) that mediate RNAito inhibit expression of two or more genes that are down-regulated byone or more miR-34 family member.

The siNA agents discussed herein include otherwise unmodified RNA aswell as RNAs which have been chemically modified, e.g., to improveefficacy, and polymers of nucleoside surrogates. Unmodified RNA refersto a molecule in which the components of the nucleic acid, namelysugars, bases, and phosphate moieties, are the same or essentially thesame as that which occur in nature, preferably as occur naturally in thehuman body. The art has referred to rare or unusual, but naturallyoccurring, RNAs as modified RNAs, see, e.g., Limbach et al., 1994,Nucleic Acids Res. 22:2183-2196. Such rare or unusual RNAs, often termedmodified RNAs (apparently because they are typically the result of apost-transcriptional modification) are within the term unmodified RNA,as used herein.

Modified RNA as used herein refers to a molecule in which one or more ofthe components of the nucleic acid, namely sugars, bases, and phosphatemoieties that are the components of the RNAi duplex, are different fromthat which occur in nature, preferably different from that which occursin the human body. While they are referred to as “modified RNAs,” theywill of course, because of the modification, include molecules which arenot RNAs. Nucleoside surrogates are molecules in which the ribophosphatebackbone is replaced with a non-ribophosphate construct that allows thebases to be presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of all of the above are discussed herein.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., ansiNA construct. It may be desirable to modify one or both of theantisense and sense strands of an siNA construct. As nucleic acids arepolymers of subunits or monomers, many of the modifications describedbelow occur at a position which is repeated within a nucleic acid, e.g.,a modification of a base, or a phosphate moiety, or the non-linking O ofa phosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid, but in many, and in fact inmost, cases it will not.

By way of example, a modification may occur at a 3′ or 5′ terminalposition, may occur in a terminal region, e.g. at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. For example, a phosphorothioate modificationat a non-linking O position may only occur at one or both termini, mayonly occur in a terminal region, e.g., at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, ormay occur in double strand and single strand regions, particularly attermini. Similarly, a modification may occur on the sense strand,antisense strand, or both. In some cases, a modification may occur on aninternal residue to the exclusion of adjacent residues. In some cases,the sense and antisense strands will have the same modifications, or thesame class of modifications, but in other cases the sense and antisensestrands will have different modifications, e.g., in some cases it may bedesirable to modify only one strand, e.g., the sense strand. In somecases, the sense strand may be modified, e.g., capped in order topromote insertion of the anti-sense strand into the RISC complex.

Other suitable modifications that can be made to a sugar, base, orbackbone of an siNA construct are described in US2006/0217331,US2005/0020521, WO2003/70918, WO2005/019453, PCT Application No.PCT/US2004/01193. An siNA construct can include a non-naturallyoccurring base, such as the bases described in any one of the abovementioned references. See also PCT Application No. PCT/US2004/011822. AnsiNA construct can also include a non-naturally occurring sugar, such asa non-carbohydrate cyclic carrier molecule. Exemplary features ofnon-naturally occurring sugars for use in siNA agents are described inPCT Application No. PCT/US2004/11829.

Two prime objectives for the introduction of modifications into siNAconstructs of the invention is their stabilization towards degradationin biological environments and the improvement of pharmacologicalproperties, e.g., pharmacodynamic properties. There are several examplesin the art describing sugar, base and phosphate modifications that canbe introduced into nucleic acid molecules with significant enhancementin their nuclease stability and efficacy. For example, oligonucleotidesare modified to enhance stability and/or enhance biological activity bymodification with nuclease resistant groups, for example, 2′-amino,2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide basemodifications (for a review see Usman and Cedergren, 1992, TIBS 17:34;Usman et al., 1994, Nucleic Acids Symp. Ser. 31:163; Burgin et al.,1996, Biochemistry, 35:14090). Sugar modification of nucleic acidmolecules has been extensively described in the art (see Eckstein etal., International Publication PCT No. WO 92/07065; Perrault et al.,1990, Nature, 344:565-568; Pieken et al., 1991, Science 253:314-317;Usman and Cedergren, 1992, Trends in Biochem. Sci. 17:334-339; Usman etal. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No.5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270:25702;Beigelman et al., International PCT publication No. WO 97/26270;Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No.5,627,053; Woolf et al., International PCT Publication No. WO 98/13526;Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20,1998; Karpeisky et al., 1998, Tetrahedron Lett., 39:1131; Earnshaw andGait, 1998, Biopolymers (Nucleic Acid Sciences), 48:39-55; Verma andEckstein, 1998, Annu. Rev. Biochem., 67:99-134; and Burlina et al.,1997, Bioorg. Med. Chem., 5:1999-2010). Such publications describegeneral methods and strategies to determine the location ofincorporation of sugar, base, and/or phosphate modifications and thelike, into nucleic acid molecules without modulating catalysis. In viewof such teachings, similar modifications can be used as described hereinto modify the siNA molecules of the instant invention so long as theability of siNA to promote RNAi in cells is not significantly inhibited.

Modifications may be modifications of the sugar-phosphate backbone.Modifications may also be modifications of the nucleoside portion.Optionally, the sense strand is an RNA or RNA strand comprising 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% modified nucleotides. Inone embodiment, the sense polynucleotide is an RNA strand comprising aplurality of modified ribonucleotides. Likewise, in other embodiments,the RNA antisense strand comprises one or more modifications. Forexample, the RNA antisense strand may comprise no more than 5%, 10%,20%, 30%, 40%, 50%, or 75% modified nucleotides. The one or moremodifications may be selected so as to increase the hydrophobicity ofthe double-stranded nucleic acid, in physiological conditions, relativeto an unmodified double-stranded nucleic acid having the same designatedsequence.

In certain embodiments, the siNA construct comprising the one or moremodifications has a log P value at least 0.5 log P units less than thelog P value of an otherwise identical unmodified siRNA construct. Inanother embodiment, the siNA construct comprising the one or moremodifications has at least 1, 2, 3, or even 4 log P units less than thelog P value of an otherwise identical unmodified siRNA construct. Theone or more modifications may be selected so as to increase the positivecharge (or increase the negative charge) of the double-stranded nucleicacid, in physiological conditions, relative to an unmodifieddouble-stranded nucleic acid having the same designated sequence. Incertain embodiments, the siNA construct comprising the one or moremodifications has an isoelectric pH (pI) that is at least 0.25 unitshigher than the otherwise identical unmodified siRNA construct. Inanother embodiment, the sense polynucleotide comprises a modification tothe phosphate-sugar backbone selected from the group consisting of: aphosphorothioate moiety, a phosphoramidate moiety, a phosphodithioatemoiety, a PNA moiety, an LNA moiety, a 2′-O-methyl moiety, and a2′-deoxy-2′ fluoride moiety.

In certain embodiments, the RNAi construct is a hairpin nucleic acidthat is processed to an siRNA inside a cell. Optionally, each strand ofthe double-stranded nucleic acid may be 19-100 base pairs long, andpreferably 19-50 or 19-30 base pairs long.

An siNAi construct can include an internucleotide linkage (e.g., thechiral phosphorothioate linkage) useful for increasing nucleaseresistance. In addition, or in the alternative, an siNA construct caninclude a ribose mimic for increased nuclease resistance. Exemplaryinternucleotide linkages and ribose mimics for increased nucleaseresistance are described in PCT Application No. PCT/US2004/07070.

An siRNAi construct can also include ligand-conjugated monomer subunitsand monomers for oligonucleotide synthesis. Exemplary monomers aredescribed, for example, in U.S. application Ser. No. 10/916,185.

An siNA construct can have a ZXY structure, such as is described inco-owned PCT Application No. PCT/US2004/07070. Likewise, an siNAconstruct can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with siNA agents are described in PCTApplication No. PCT/US2004/07070.

The sense and antisense sequences of an siNAi construct can bepalindromic. Exemplary features of palindromic siNA agents are describedin PCT Application No. PCT/US2004/07070.

In another embodiment, the siNA construct of the invention can becomplexed to a delivery agent that features a modular complex. Thecomplex can include a carrier agent linked to one or more of (preferablytwo or more, more preferably all three of): (a) a condensing agent(e.g., an agent capable of attracting, e.g., binding, a nucleic acid,e.g., through ionic or electrostatic interactions); (b) a fusogenicagent (e.g., an agent capable of fusing and/or being transported througha cell membrane); and (c) a targeting group, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g.,an antibody, that binds to a specified cell type. iRNA agents complexedto a delivery agent are described in PCT Application No.PCT/US2004/07070.

The siNA construct of the invention can have non-canonical pairings,such as between the sense and antisense sequences of the iRNA duplex.Exemplary features of non-canonical iRNA agents are described in PCTApplication No. PCT/US2004/07070.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample, Lin and Matteucci, 1998, J. Am. Chem. Soc., 120:8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication Nos. WO00/66604 and WO 99/14226).

An siNA agent of the invention can be modified to exhibit enhancedresistance to nucleases. An exemplary method proposes identifyingcleavage sites and modifying such sites to inhibit cleavage. Anexemplary dinucleotide 5′-UA-3′,5′-UG-3′,5′-CA-3′,5′-UU-3′, or 5′-CC-3′as disclosed in PCT/US2005/018931 may serve as a cleavage site.

For increased nuclease resistance and/or binding affinity to the target,a siRNA agent, e.g., the sense and/or antisense strands of the iRNAagent, can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl, aralkyl,heteroaryl, or sugar); polyethyleneglycols (PEG),O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon ofthe same ribose sugar; O-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino, ethylene diamine, polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE,(e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino,diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino). It is noteworthy that oligonucleotides containing only themethoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibitnuclease stabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e., deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g., NH₂, alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with, e.g., an amino functionality. In oneembodiment, the substituents are 2′-methoxyethyl, 2′-OCH₃, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

In another embodiment, to maximize nuclease resistance, the 2′modifications may be used in combination with one or more phosphatelinker modifications (e.g., phosphorothioate). The so-called “chimeric”oligonucleotides are those that contain two or more differentmodifications.

In certain embodiments, all the pyrimidines of a siNA agent carry a2′-modification, and the molecule therefore has enhanced resistance toendonucleases. Enhanced nuclease resistance can also be achieved bymodifying the 5′ nucleotide, resulting, for example, in at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide; at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide; or at least one 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide. The siNA agent can include at least 2, at least 3, at least4 or at least 5 of such dinucleotides. In some embodiments, the 5′-mostpyrimidines in all occurrences of the sequence motifs 5′-UA-3′,5′-CA-3′,5′-UU-3′, and 5′-UG-3′ are 2′-modified nucleotides. In otherembodiments, all pyrimidines in the sense strand are 2′-modifiednucleotides, and the 5′-most pyrimidines in all occurrences of thesequence motifs 5′-UA-3′ and 5′-CA-3′. In one embodiment, allpyrimidines in the sense strand are 2′-modified nucleotides, and the5′-most pyrimidines in all occurrences of the sequence motifs5′-UA-3′,5′-CA-3′,5′-UU-3′, and 5′-UG-3′ are 2′-modified nucleotides inthe antisense strand. The latter patterns of modifications have beenshown to maximize the contribution of the nucleotide modifications tothe stabilization of the overall molecule towards nuclease degradation,while minimizing the overall number of modifications required to achievea desired stability, see PCT/US2005/018931. Additional modifications toenhance resistance to nucleases may be found in US2005/0020521,WO2003/70918, and WO2005/019453.

The inclusion of furanose sugars in the oligonucleotide backbone canalso decrease endonucleolytic cleavage. Thus, in one embodiment, thesiNA of the invention can be modified by including a 3′ cationic group,or by inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage.In another alternative, the 3′-terminus can be blocked with anaminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates caninhibit 3′-5′ exonucleolytic cleavage. While not being bound by theory,a 3′ conjugate, such as naproxen or ibuprofen, may inhibitexonucleolytic cleavage by sterically blocking the exonuclease frombinding to the 3′-end of oligonucleotide. Even small alkyl chains, arylgroups, heterocyclic conjugates, or modified sugars (D-ribose,deoxyribose, glucose, etc.) can block 3′-5′-exonucleases.

Similarly, 5′ conjugates can inhibit 5′-3′ exonucleolytic cleavage.While not being bound by theory, a 5′ conjugate, such as naproxen oribuprofen, may inhibit exonucleolytic cleavage by sterically blockingthe exonuclease from binding to the 5′-end of oligonucleotide. Evensmall alkyl chains, aryl groups, heterocyclic conjugates, or modifiedsugars (D-ribose, deoxyribose, glucose, etc.) can block3′-5′-exonucleases.

An alternative approach to increasing resistance to a nuclease by ansiNA molecule proposes including an overhang to at least one or bothstrands of a duplex siNA. In some embodiments, the nucleotide overhangincludes 1 to 4, preferably 2 to 3, unpaired nucleotides. In anotherembodiment, the unpaired nucleotide of the single-stranded overhang thatis directly adjacent to the terminal nucleotide pair contains a purinebase, and the terminal nucleotide pair is a G-C pair, or at least two ofthe last four complementary nucleotide pairs are G-C pairs. In otherembodiments, the nucleotide overhang may have 1 or 2 unpairednucleotides, and in an exemplary embodiment the nucleotide overhang maybe 5′-GC-3′. In another embodiment, the nucleotide overhang is on the3′-end of the antisense strand.

Thus, an siNA molecule can include monomers which have been modified soas to inhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These monomers arereferred to herein as NRMs, or Nuclease Resistance promoting Monomers ormodifications. In some cases these modifications will modulate otherproperties of the siNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC, or the ability of the first and second sequences to form aduplex with one another or to form a duplex with another sequence, e.g.,a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an siNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. In some embodiments, themodification may increase exonuclease and endonuclease resistance andthus prolong the half-life of the siNA agent prior to interaction withthe RISC complex, but at the same time does not render the siNA agentinactive with respect to its intended activity as a target mRNA cleavagedirecting agent. Again, while not wishing to be bound by any theory, itis believed that placement of the modifications at or near the 3′ and/or5′-end of antisense strands can result in siNA agents that meet thepreferred nuclease resistance criteria delineated above.

Modifications that can be useful for producing siNA agents that exhibitthe nuclease resistance criteria delineated above may include one ormore of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone, it being understood that theart discloses other methods as well that can achieve the same result:

(i) chiral (Sp) thioates. An NRM may include nucleotide dimers enrichedor pure for a particular chiral form of a modified phosphate groupcontaining a heteroatom at the nonbridging position, e.g., Sp or Rp, atthe position X, where this is the position normally occupied by theoxygen. The atom at X can also be S, Se, Nr₂, or Br₃. When X is S,enriched or chirally pure Sp linkage is preferred. Enriched means atleast 70, 80, 90, 95, or 99% of the preferred form.

(ii) attachment of one or more cationic groups to the sugar, base,and/or the phosphorus atom of a phosphate or modified phosphate backbonemoiety. In some embodiments, these may include monomers at the terminalposition derivatized at a cationic group. As the 5′-end of an antisensesequence should have a terminal —OH or phosphate group, this NRM ispreferably not used at the 5′-end of an antisense sequence. The groupshould preferably be attached at a position on the base which minimizesinterference with H bond formation and hybridization, e.g., away fromthe face which interacts with the complementary base on the otherstrand, e.g., at the 5′ position of a pyrimidine or a 7-position of apurine.

(iii) nonphosphate linkages at the termini. In some embodiments, theNRMs include non-phosphate linkages, e.g., a linkage of 4 atoms whichconfers greater resistance to cleavage than does a phosphate bond.Examples include 3′ CH₂—NCH₃—O—CH₂-5′ and 3′ CH₂—NH—(O.dbd.)-CH₂-5′.

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Incertain embodiments, the NRMs can be included among these structures.

(v) L-RNA, 2′-5′ linkages, inverted linkages, and a-nucleosides. Incertain embodiments, the NRMs include: L nucleosides and dimericnucleotides derived from L-nucleosides; 2′-5′ phosphate, non-phosphateand modified phosphate linkages (e.g., thiophosphates, phosphoramidatesand boronophosphates); dimers having inverted linkages, e.g., 3′-3′ or5′-5′ linkages; monomers having an alpha linkage at the 1′ site on thesugar, e.g., the structures described herein having an alpha linkage,

(vi) conjugate groups. In certain embodiments, the NRMs can include,e.g., a targeting moiety or a conjugated ligand described hereinconjugated with the monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. In certain embodiments, the NRMs can include anabasic monomer, e.g., an abasic monomer as described herein (e.g., anucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclicaromatic monomer as described herein; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. In certain embodiments,the NRMs include monomers, preferably at the terminal position, e.g.,the 5′ position, in which one or more atoms of the phosphate group isderivatized with a protecting group, which protecting group or groupsare removed as a result of the action of a component in the subject'sbody, e.g., a carboxyesterase or an enzyme present in the subject'sbody. For example, a phosphate prodrug in which a carboxy esterasecleaves the protected molecule resulting in the production of a thioateanion which attacks a carbon adjacent to the 0 of a phosphate andresulting in the production of an unprotected phosphate.

“Ligand,” as used herein, means a molecule that specifically binds to asecond molecule, typically a polypeptide or portion thereof, such as acarbohydrate moiety, through a mechanism other than an antigen-antibodyinteraction. The term encompasses, for example, polypeptides, peptides,and small molecules, either naturally occurring or synthesized,including molecules whose structure has been invented by man. Althoughthe term is frequently used in the context of receptors and moleculeswith which they interact and that typically modulate their activity(e.g., agonists or antagonists), the term as used herein applies moregenerally.

One or more different NRM modifications can be introduced into a siNAagent or into a sequence of a siRNA agent. An NRM modification can beused more than once in a sequence or in a siRNA agent. As some NRMsinterfere with hybridization, the total number incorporated should besuch that acceptable levels of siNA agent duplex formation aremaintained.

In some embodiments, NRM modifications are introduced into the terminalcleavage site or in the cleavage region of a sequence (a sense strand orsequence) which does not target a desired sequence or gene in thesubject.

In most cases, the nuclease-resistance promoting modifications will bedistributed differently depending on whether the sequence will target asequence in the subject (often referred to as an antisense sequence) orwill not target a sequence in the subject (often referred to as a sensesequence). If a sequence is to target a sequence in the subject,modifications which interfere with or inhibit endonuclease cleavageshould not be inserted in the region which is subject to RISC mediatedcleavage, e.g., the cleavage site or the cleavage region (as describedin Elbashir et al., 2001, Genes and Dev. 15:188). Cleavage of the targetoccurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11nucleotides upstream of the first nucleotide which is complementary tothe guide sequence. As used herein, “cleavage site” refers to thenucleotide on either side of the cleavage site, on the target, or on theiRNA agent strand which hybridizes to it. Cleavage region means anucleotide within 1, 2, or 3 nucleotides of the cleavage site, in eitherdirection.

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position, or within 2, 3, 4, or 5 positions of theterminus, of a sequence which targets or a sequence which does nottarget a sequence in the subject.

V. THERAPEUTIC USE

Tumors having a defective TP53 pathway status are hypothesized to bemore responsive to several oncology compounds in development (PLK1,AURA, WEE1, CHEK1) (WO 2005031002). Therefore, identification oftranscripts that predict TP53 functional status may be useful for theselection of appropriate patient populations for clinical testing ofthese compounds. Previous studies have used genome-scale approaches toidentify transcriptional markers for TP53 function. Chromatinimmunoprecipitation (ChIP) was used for genome-scale analysis of TP53transcription factor binding sites (Wie et al., (2006) Cell 124:207-219)Miller et al. analyzed breast cancers with sequenced TP53 and identifiedan expression signature that distinguished TP53-mutant and wild-typetumors, and predicted therapeutic responses (Miller et al., (2005) PNAS102:13550-13555).

In one embodiment, a method is provided for treating a mammalian subjecthaving a cancer, comprising (a) classifying a cancer cell sample fromthe subject as having an active TP53 pathway or an inactive TP53pathway; and (b) treating a mammalian subject having an active TP53pathway with a composition comprising a small interfering nucleic acid(siNA), wherein said siNA comprises a guide strand contiguous nucleotidesequence of at least 18 nucleotides, wherein said guide strand comprisesa seed region consisting of nucleotide positions 1 to 12, whereinposition 1 represents the 5′ end of said guide strand and wherein saidseed region comprises a nucleotide sequence of at least six contiguousnucleotides that is identical to six contiguous nucleotides within asequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO:9, and SEQ ID NO:31.

Examples of cancers that can be treated using the compositions of theinvention include, but are not limited to: biliary tract cancer; bladdercancer; brain cancer including glioblastomas and medulloblastomas;breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; hematologicalneoplasms including acute lymphocytic and myelogenous leukemia; multiplemyeloma; AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastomas; oral cancer including squamouscell carcinoma; ovarian cancer including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreaticcancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, andosteosarcoma; skin cancer including melanoma, Kaposi's sarcoma,basocellular cancer, and squamous cell cancer; testicular cancerincluding germinal tumors such as seminoma, non-seminoma, teratomas,choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancerincluding thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms' tumor.

In some embodiments, the compositions of the invention comprising asmall interfering nucleic acid (siNA) are used to treat mammaliansubjects afflicted with commonly encountered cancers such as breast,prostate, lung, ovarian, colorectal, and brain cancer. In someembodiments, the compositions of the invention comprising a smallinterfering nucleic acid (siNA) are used to inhibit the proliferation ofa cancer cell that is c-MET dependent. In some embodiments, thecompositions of the invention are used to treat mammalina subjectsafflicted with c-MET dependent non-small cell lung carcinoma.

In general, an effective amount of the one or more compositions of theinvention for treating a mammalian subject afflicted with cancer will bethat amount necessary to inhibit mammalian cancer cell proliferation insitu. Those of ordinary skill in the art are well-schooled in the art ofevaluating effective amounts of anti-cancer agents.

In some cases, the above-described treatment methods may be combinedwith known cancer treatment methods. The term “cancer treatment” as usedherein, may include, but is not limited to, chemotherapy, radiotherapy,adjuvant therapy, surgery, or any combination of these and/or othermethods. Particular forms of cancer treatment may vary, for instance,depending on the subject being treated. Examples include, but are notlimited to, dosages, timing of administration, duration of treatment,etc. One of ordinary skill in the medical arts can determine anappropriate cancer treatment for a subject.

The molecules of the instant invention can be used as pharmaceuticalagents. Pharmaceutical agents prevent, inhibit the occurrence of, ortreat (alleviate a symptom to some extent, preferably all of thesymptoms) a disease state in a subject.

The negatively charged polynucleotides of the invention can beadministered (e.g., RNA, DNA or protein complex thereof) and introducedinto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration; suppositories for rectaladministration; sterile solutions; suspensions for injectableadministration; and the other compositions known in the art.

In some embodiments, the compositions of the present invention areadministered locally to a localized region of a subject, such as atumor, via local injection.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, preferably a human. Suitableforms, in part, depend upon the use or the route of entry, for exampleoral, transdermal, or by injection. Such forms should not prevent thecomposition or formulation from reaching a target cell (i.e., a cell towhich the negatively charged polymer is desired to be delivered). Forexample, pharmacological compositions injected into the blood streamshould be soluble. Other factors are known in the art, and includeconsiderations such as toxicity and forms which prevent the compositionor formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes which lead to systemicabsorption include, without limitations: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular.Each of these administration routes exposes the desired negativelycharged polymers, e.g., nucleic acids, to an accessible diseased tissue.The rate of entry of a drug into the circulation has been shown to be afunction of molecular weight or size. The use of a liposome or otherdrug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation which can facilitate the association of drug withthe surface of cells, such as lymphocytes and macrophages, is alsouseful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: PEG conjugated nucleic acids, phospholipid conjugatednucleic acids, nucleic acids containing lipophilic moieties,phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85)which can enhance entry of drugs into various tissues, for example theCNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol.,13:16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide)microspheres for sustained release delivery after implantation (Emerich,D. F. et al., 1999, Cell Transplant, 8:47-58) Alkermes, Inc. Cambridge,Mass.; and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23:941-949, 1999). Nanoparticlesfunctionalized with lipids (lipid nanoparticles), such aslysine-containing nanoparticles with the surface functional groupsmodified with lipid chains may also be used for delivery of the nucleicacid molecules of the instant invention. Such lipid nanoparticles may begenerated as described in Baigude H. et al., ACS Chemical Biology Vol2(4):237-241 (2007), incorporated herein by reference. Othernon-limiting examples of delivery strategies, including CNS delivery ofthe nucleic acid molecules of the instant invention include materialdescribed in Boado et al., 1998, J. Pharm. Sci., 87:1308-1315; Tyler etal., 1999, FEBS Lett., 421:280-284; Pardridge et al., 1995, PNAS USA.,92:5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15:73-107;Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26:4910-4916; andTyler et al., 1999, PNAS USA., 96:7053-7058. All these references arehereby incorporated herein by reference.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).Nucleic acid molecules of the invention can also comprise covalentlyattached PEG molecules of various molecular weights. These formulationsoffer a method for increasing the accumulation of drugs in targettissues. This class of drug carriers resists opsonization andelimination by the mononuclear phagocytic system (MPS or RES), therebyenabling longer blood circulation times and enhanced tissue exposure forthe encapsulated drug (Lasic et al., 1995, Chem. Rev. 95:2601-2627;Ishiwata et al., 1995, Chem. Pharm. Bull. 43:1005-1011). Such liposomeshave been shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., 1995, Science 267:1275-1276; Oku et al., 1995, Biochim. Biophys.Acta, 1238:86-90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., 1995, J. Biol. Chem.42:24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392; all ofwhich are incorporated by reference herein). Long-circulating liposomesare also likely to protect drugs from nuclease degradation to a greaterextent compared to cationic liposomes, based on their ability to avoidaccumulation in metabolically aggressive MPS tissues such as the liverand spleen. All of these references are incorporated by referenceherein. The present invention also includes compositions prepared forstorage or administration which include a pharmaceutically effectiveamount of the desired compounds in a pharmaceutically acceptable carrieror diluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroed., 1985) hereby incorporated by reference herein. For example,preservatives, stabilizers, dyes, and flavoring agents can be provided.These include sodium benzoate, sorbic acid, and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentscan be used.

A pharmaceutically effective dose is the dose required to prevent,inhibit the occurrence of, or treat (alleviate a symptom to some extent,preferably all of the symptoms) a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors which those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered, depending uponthe potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.The term parenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising a nucleic acid moleculeof the invention and a pharmaceutically acceptable carrier. One or morenucleic acid molecules of the invention can be present in associationwith one or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants, and, if desired, other active ingredients.The pharmaceutical compositions containing nucleic acid molecules of theinvention can be in a form suitable for oral use, for example, astablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs.

Compositions intended for oral use can be prepared according to anymethod known in the art for the manufacture of pharmaceuticalcompositions, and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents, or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch or alginic acid; binding agents, for examplestarch, gelatin, or acacia, and lubricating agents, for examplemagnesium stearate, stearic acid, or talc. The tablets can be uncoatedor they can be coated by known techniques. In some cases such coatingscan be prepared by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients may include suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents such as a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil, orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring, and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example, gum acacia or gum tragacanth;naturally-occurring phosphatides, for example, soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol;anhydrides, for example, sorbitan monooleate; and condensation productsof the said partial esters with ethylene oxide, for example,polyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol, glucose, or sucrose. Suchformulations can also contain a demulcent, a preservative, and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels on the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient orsubject per day). The amount of active ingredient that can be combinedwith the carrier materials to produce a single dosage form variesdepending upon the host treated and the particular mode ofadministration. Dosage unit forms generally contain between from about 1mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular patientor subject depends upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination, and the severity of the particular diseaseundergoing therapy.

For administration to non-human animals, the composition can also beadded to the animal's feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

Examples are provided below to further illustrate different features andadvantages of the present invention. The examples also illustrate usefulmethodology for practicing the invention. These examples do not limitthe claimed invention.

Example 1

This Example demonstrates that shRNA-mediated suppression of TP53downregulates expression of an EST Cluster (Contig6654) that containsthe miR-34a locus.

Rationale:

Cells having wild type TP53 arrest at a G1 checkpoint following DNAdamage to allow DNA repair prior to cell cycle progression.shRNA-mediated disruption of TP53 eliminates this G1 arrest (Brummelkampet al., (2002) Science 296:550-553). A series of tumor cell lines weretested for G1 arrest following Doxorubicin treatment to confirm theintegrity of the TP53 pathway. Eight tumor lines reported as havingnormal TP53 activity were used in this study: A549 (lung carcinoma,O'Connor et al., Cancer Res. 57:4285-300), TOV21G (ovarian carcinoma,Samouelian et al., 2004, Cancer Chemother. Pharmacol 54:497-504), MCF7(breast carcinoma, Concin et al., 2003 Breast Cancer Res. Treat.79:37-46), HEPG2 (hepatic carcinoma, Bamford, et al., 2004, Br. J.Cancer 91:355-58), OAW42 (ovarian carcinoma, Bamford et al., 2004, Br.J. Cancer 91:355-58); A2780 (ovarian carcinoma, Bamford, et al., 2004,Br. J. Cancer 91:355-58); U2OS (osteosarcoma, Zhu et al., 1993 Genes &Dev. 7:1111-25); and NCI-H460 (lung carcinoma, O'Connor et al., 1997Cancer Res. 57:4285-300).

Methods:

A series of matched cell line pairs with or without functional TP53 werecreated. Multiple cell lines were made to avoid idiosyncratic effectsparticular to any single cell line. Stable cell lines were transducedwith an empty lentiviral vector or with a lentiviral vector encoding anshRNA targeting TP53. The vectors used were pLenti6/BLOCK-iT-DESTdestination vectors (Invitrogen Corporation, Carlsbad, Calif.) intowhich had been transferred a Gateway (Invitrogen)-compatible expressioncassette containing the human H1 promoter upstream of an shRNA targetingTP53 or a terminator sequence consisting of a stretch of fivethymidines, a BamHI site, and then another five thymidines.

TP53 shRNA used in these experiments had the 19-nucleotide core sequence5′ GACUCCAGUGGUAAUCUAC 3′ [SEQ ID NO:10]. The full hairpin sequencecloned into the lentiviral vector was: 5′ GACUCCAGUGGUAAUCUACUUCAAGAGAGUAGAUUACCACUGGAGUCUUUUU 3′ [SEQ ID NO:11].

TP53 mRNA levels were reduced by ˜80-95% in cell lines expressing theTP53 shRNA as compared with cells transduced with empty vector (data notshown). A549 cells (lung carcinoma) were transduced with an emptylentiviral vector (LV vector) or with a vector encoding an shRNA hairpintargeting TP53 (p53 shRNA), and stable cell lines were isolated. Inbrief, cells at 50% to 70% confluence were inoculated with virus at amultiplicity of infection (MOI) of 10 transducing units per cell(TU/cell) in DMEM with 10% FBS and 6 μg/ml polybrene. After 24 hours,the virus was removed and the cultures were replenished with fresh DMEMplus 10% FBS. Transduced cells were drug selected with 5 ug/mlblasticidin, which was added to the medium 4-5 days after transduction.Stable cells were treated with doxorubicin (+Doxorubicin) or without(Doxorubicin) for 24 hours and then subjected to cell cycle analysis byflow cytometry.

As shown in FIGS. 2A-2D, all cells expressing TP53 shRNA showed reducedG1 arrest following treatment with doxorubicin to induce DNA damage.FIG. 2A is a histogram of cells with wildtype p53 showing the number ofcells (Y axis) with a given DNA content (measured by fluorescenceintensity, X axis). FIG. 2B is a histogram of cells with wildtype p53treated with doxorubicin showing the number of cells (Y axis) with agiven DNA content (measured by fluorescence intensity, X axis). FIG. 2Cis a histogram of cells with wildtype p53 transfected with TP53 shRNAshowing the number of cells (Y axis) with a given DNA content (measuredby fluorescence intensity, X axis), and FIG. 2D is a histogram of cellswith wildtype p53 transfected with TP53 shRNA and treated withdoxorubicin showing the number of cells (Y axis) with a given DNAcontent (measured by fluorescence intensity, X axis), showing thatdisruption of TP53 ablates G0/G1 checkpoint following DNA damage. Asshown in FIGS. 2A-2D, suppression of TP53 diminishes the G0/G1checkpoint.

Messenger RNA (mRNA) was isolated from each line and subjected to DNAmicroarray analysis, with comparisons made between cells transduced withempty Lentivirus vector versus cells transduced with the Lentivirusvector encoding TP53 shRNA. To eliminate experimental noise, genes wereidentified as being regulated by TP53 if they were regulated >1.5-fold,P<0.01, in 5 or more cell lines. On the basis of these criteria,Contig6654 was identified as a transcript that was affected by the TP53shRNA disruption of TP53 function.

Table 2 provides the fold change in log 10 ratio of hybridizationintensity for the selected genes in empty vector-transduced cellscompared with TP53 shRNA-transduced A549 cells. Results shown in Table 2are derived from competitive hybridization microarray studies comparingA549 cells expressing a TP53 targeting shRNA versus A549 cells carryinga control vector. TP53 was down-regulated in the about 5-fold in cellsexpressing the shRNA targeting TP53 verses cells expressing an emptyvector.

TABLE 2 Effects of shRNA-mediated suppression of TP53 on transcriptlevels of known TP53 regulated genes. Primary Sequence Name Accession #Fold change in transcript level TP53I3 NM_004881 1.153201 INPP5DNM_005541 −1.650142 DDB2 NM_000107 −1.853958 CYFIP2 NM_030778 −2.091289CDKN1A NM_000389 −2.623143 TRIM22 NM_006074 −1.203311 ACTA2 NM_001613−3.774972 FAS NM_152873 −1.93776 BTG2 NM_006763 −1.828167 SESN1NM_014454 −2.000987 FDXR NM_004110 −2.132721 BBC3 NM_014417 −1.516828TP53INP1 NM_033285 −3.438426 PLK2 NM_006622 −1.067533 PHLDA3 NM_012396−1.722152 RRM2B NM_015713 −1.806952 GADD45A NM_001924 −1.288047 BAXNM_138763 1.010626 INSIG1 NM_005542 −1.579872 Contig6654_RC −1.936355

Each cell line pair gave distinct but overlapping gene expressionsignatures, primarily comprising low magnitude regulations. As shown inTable 2, TP53 was strongly down-regulated in all cases, but most of theother reporters showed weaker regulations that varied between differentcell lines.

Contig6654_RC is a poorly characterized EST cluster. Mapping of thiscontig to the human genome was performed using a genome browser softwareand database package publicly provided by the University of Californiaat Santa Cruz (UCSC) which included a comparison of STS Markers ongenetic and radiation hybrid maps, known genes based on UniProt, RefSeqand GenBank mRNA and microRNA species as described in the publiclyavailable miRBase sequence database as described in Griffith-Jones etal., Nucleic Acids Research 32:D109-D111 (2004) and Griffith-Jones etal., Nucleic Acids Research 34:D140-D144 (2006), accessible on the WorldWide Web at the Wellcome Trust Sanger Institute website. Inspection ofthe above described information in the UCSC genome browser revealed thatContig6654 belongs to an EST cluster that overlaps with a microRNAlocus, miR-34a, leading to the hypothesis that disruption of TP53function down-regulates a miRNA precursor of miR-34a.

Example 2

This Example demonstrates that the introduction of synthetic miR-34 intohuman cells elicits a phenotype similar to that induced by activation ofthe TP53 G1 checkpoint.

Rationale:

Delay of the G1/S transition of the cell cycle is known to be aconsequence of TP53 activation. In this example, miR-34 siRNA duplexeswere designed with passenger strands that are complementary to thenatural mature miRNA, except for a single base mismatch four bases fromthe 3′ end of the sequence, referred to as “asymmetric passengerstrands”. Exemplary asymmetric passenger strands are provided in Table 3for miR-34a (SEQ ID NO:12); for miR-34b (SEQ ID NO:17), and for miR-34c(SEQ ID NO:22), with the mismatch underlined. As shown in Table 3, thesesynthetically designed asymmetric passenger strands differ from thecorresponding natural passenger strands for miR-34a (SEQ ID NO:35),miR-34b (SEQ ID NO:36) and miR-34c (SEQ ID NO:37).

As described in Example 5, it was determined that an siRNA duplex miR-34mimetic sequence containing annealed strands of SEQ ID NO:1 andasymmetric passenger strand SEQ ID NO:12 was more effective in inducinga cell death phenotype than annealed natural miR-34 guide strand (SEQ IDNO:1) and natural miR-34 passenger strand (SEQ ID NO:35). While notwishing to be bound by theory, it is believed that the presence of themismatch in the passenger strand destabilizes the duplex in that regionand thereby facilitates entry into RISC of the strand mimicking maturemiR-34. The duplex miR-34 mimetic sequence with the asymmetric passengerstrand and natural guide strand is processed resulting in formation ofthe mature wild type miR-34 guide strand.

The data presented in this example show that introduction of such duplexmiR-34 mimetics into cells leads to cell cycle arrest at the G1checkpoint in a manner that is analogous to activation of TP53.

Methods:

A549 cells were transfected with synthetic miR-34a, b, and c syntheticRNA duplexes, as well as mutated control versions of each. Twenty fourhours post transfection, the cells were treated with Nocodazole (100ng/ml) for 16-20 hours. The percentage of cells arrested at the G1 stageof the cell cycle was measured using propidium iodide staining and flowcytometry. All synthetic oligonucleotides (Table 3) were obtained fromSigma-Proligo (St. Louis, Mo.).

TABLE 3 Synthetic miR-34 Oligonucleotide Sequences siRNA, miRNA or SEQSEQ mismatch Guide strand/mature ID Passenger strand ID miRNA (5′ to 3′)NO: (5′ to 3′) NO: miR-34a UGGCAGUGUCUUAGCUGGUUGU 1CAAUCAGCAAGUAUACUGCCCU 35 (natural) (natural) miR-34aUGGCAGUGUCUUAGCUGGUUGU 1 AACCAGCUAAGACACUGCGAAU 12 (natural)(synthetic: reverse complement of natural guide strand withone base mismatch) miR-34a- UCCCAGUGUCUUAGCUGGUUGU 13AACCAGCUAAGACACUGGCAAU 14 mm2,3 (mutation in seed (synthetic: reverse complement region) of seed region mutation withone base mismatch) miR-34a- UGGCAGUGUCUUAGCUGCAUGU 15AUGCAGCUAAGACACUGCGAAU 16 mm18,19 (mutation in non-seed (synthetic: reverse complement region) of non-seed region mutationwith one base mismatch) miR-34b AGGCAGUGUCAUUAGCUGAUUG 4CAAUCACUAACUCCACUGCCAU 36 (natural) (natural) miR-34bAGGCAGUGUCAUUAGCUGAUUG 4 AUCAGCUAAUGACACUGCGUAU 17 (natural)(synthetic: reverse complement of natural guide strand withone base mismatch) miR-34b- ACCCAGUGUCAUUAGCUGAUUG 18AUCAGCUAAUGACACUGGCUAU 19 mm2,3 (mutation in seed (synthetic: reverse complement region) of seed region mutation withone base mismatch) miR-34b- AGGCAGUGUCAUUAGCUCUUUG 20AAGAGCUAAUGACACUGCGUAU 21 mm18,19 (mutation in non-seed (synthetic: reverse complement region) of non-seed region mutationwith one base mismatch) miR-34c AGGCAGUGUAGUUAGCUGAUUG 7AAUCACUAACCACACGGCCAGG 37 (natural) (natural) miR-34cAGGCAGUGUAGUUAGCUGAUUG 7 AUCAGCUAACUACACUGCGUAU 22 (natural)(synthetic: reverse complement of natural guide strand withone base mismatch) miR-34c- ACCCAGUGUAGUUAGCUGAUUG 23AUCAGCUAACUACACUGGCUAU 24 mm2,3 (mutation in seed (synthetic: reverse complement region) of seed region mutation withone base mismatch) miR-34c- AGGCAGUGUAGUUAGCUCUUUG 25AAGAGCUAACUACACUGCGUAU 26 mm18,19 (mutation in non-seed (synthetic: reverse complement region) of non-seed region mutationwith one base mismatch)

TABLE 4 Cell Cycle Arrest in A549 Cells (wild type p53) Transfected withSynthetic miR-34 Constructs microRNA species introduced into Guidestrand/passenger % Cells A549 cells strand in G1 miR34a SEQ ID NO: 1/SEQID NO: 12 43.3% (WT mature) miR34a-mm18,19 SEQ ID NO: 15/SEQ ID NO: 1636.0% (non-seed mismatch) miR34a-mm2,3 SEQ ID NO: 13/SEQ ID NO: 14 20.4%(seed mismatch) miR34b SEQ ID NO: 4/SEQ ID NO: 17 67.7% (WT mature)miR34b-mm18,19 SEQ ID NO: 20/SEQ ID NO: 21 60.6% (non-seed mismatch)miR34b-mm2,3 SEQ ID NO: 18/SEQ ID NO: 19 19.8% (seed mismatch) miR34cSEQ ID NO: 7/SEQ ID NO: 22 67.6% (WT mature) miR34c-mm18,19 SEQ ID NO:25/SEQ ID NO: 26 60.5% (non-seed mismatch) miR34c-mm2,3 SEQ ID NO:23/SEQ ID NO: 24 21.2% (seed mismatch)

Table 4 shows A549 cells having a normal level of TP53 function (wildtype p53) that were either transfected with a normal synthetic miR-34aRNA duplex (wild type mature) comprising a guide strand [SEQ ID NO:1]and a passenger strand [SEQ ID NO:12] with a single nucleotide mismatch;transfected with a non-seed region double mutant syntheticmiR-34a(18,19) RNA duplex comprising a guide strand [SEQ ID NO:15] and apassenger strand [SEQ ID NO:16]; or transfected with a seed regiondouble mutant synthetic miR-34a(2,3) RNA duplex comprising a guidestrand [SEQ ID NO:13] and a passenger strand [SEQ ID NO:14].

Table 4 further shows A549 cells having a normal level of TP53 function(wild type p53) that were either transfected with a normal syntheticmiR-34b RNA duplex (wild type mature) comprising a guide strand [SEQ IDNO:4] and a passenger strand [SEQ ID NO:17] with a single nucleotidemismatch; transfected with a non-seed region double mutant syntheticmiR-34b(18,19) RNA duplex comprising a guide strand [SEQ ID NO:20] and apassenger strand [SEQ ID NO:21]; or transfected with a seed regiondouble mutant synthetic miR-34b(2,3) RNA duplex comprising a guidestrand [SEQ ID NO:18] and a passenger strand [SEQ ID NO:19].

Table 4 further shows A549 cells having a normal level of TP53 function(wild type p53) that were either transfected with a normal syntheticmiR-34c RNA duplex (wild type mature) comprising a guide strand [SEQ IDNO:7] and a passenger strand [SEQ ID NO:22] with a single nucleotidemismatch; transfected with a non-seed region double mutant syntheticmiR-34b(18,19) RNA duplex comprising a guide strand [SEQ ID NO:25] and apassenger strand [SEQ ID NO:26]; or transfected with a seed regiondouble mutant synthetic miR-34c(2,3) RNA duplex comprising a guidestrand [SEQ ID NO:23] and a passenger strand [SEQ ID NO:24].

The data provided in Table 4 shows that introduction of syntheticmiR-34a, miR-34b, and miR-34c RNA duplexes (wild type mature), as wellas double mutant RNA duplexes miR-34a(18,19), miR-34b(18,19), andmiR-34c(18,19), that have mutations outside of the seed region, induce aG1 cell cycle arrest in a cell having a normal level of TP53 function.RNA duplexes miR-34a(2,3), miR-34b(2,3), and miR-34c(2,3), that havedouble mutations in the seed region, do not induce such a cell cyclearrest. Thus, each of the synthetic miR-34a, miR-34b, and miR-34c siRNAconstructs that have a corresponding intact seed region, can elicit aphenotype reflective of the TP53-mediated DNA damage checkpoint.

It was also observed that the cell cycle arrest phenotype induced byintroduction of miR-34a or miR-34a(18-19), miR34b or miR34b(18-19), ormiR34c or miR34c(18-19) is dependent on TP53 function. Delivery of thesame set of miR-34a synthetic siRNA constructs (Table 3) to A549 cellsstably expressing a TP53 shRNA construct that silences TP53 to about 5%of the levels in control A549 cells did not result in the cell cyclearrest phenotype (data not shown).

Example 3

This Example demonstrates that transcripts regulated by miR-34 overlapwith TP53 pathway genes.

Methods:

To better understand the function of the miR-34 family, gene expressionprofiling experiments were performed. RNA duplexes corresponding tomiR-34a, miR-34b, miR-34c, or a control target luciferase (Luc) weretransfected into A549, HCT116 Dicer^(ex5), TOV21G, DLD-1 Dicer^(ex5)cells. The guide strand of the luciferase siRNA used in theseexperiments was: 5′ CGUACGCGGAAUACUUCGAdTdT 3′ [SEQ ID NO:27], and thepassenger strand of the luciferase siRNA was 5′ UCGAAGUAUUCCGUACGdTdT 3[SEQ ID NO:28] (purchased from Sigma-Proligo). The miR-34a, miR-34b, andmiR-34c siRNA duplexes used in these experiments are set forth in Table3 of Example 2.

HCT116 cells were transfected in 6-well plates by using Lipofectamine2000 (Invitrogen, Carlsbad, Calif.). DLD-1, TOV21G, and A549 cells weretransfected using SilentFect (Bio-Rad, Hercules, Calif.). Duplexes wereused at final concentrations of 100 nM for all cell lines. Total RNA wasisolated 24 hours post transfection, and subjected to microarrayexpression analysis as described by Jackson et al. (2003 Nat.Biotechnol. 21:635-37). Microarray profiling of cells transfected withthe miR-34a, b, and c-like siRNA sequences were used to identify thedirect targets of the miR-34 microRNAs, as well as their downstreameffects.

Results:

Analysis of the microarray gene expression profiles (data not shown)identified a cluster of genes that were specifically down-regulated at24 hours post-transfection as shown in Table 5 below. Genesdown-regulated by miR-34 were highly enriched for transcripts containing3′UTRs complementary to the miR-34 seed region hexamers.MicroRNA-regulated transcripts were identified in microarray geneexpression signatures using a P-value cut-off (P<0.01). miRNAdown-regulated transcripts were defined by the intersection ofdown-regulated transcripts in all the lines tested. Down-regulatedtranscripts were tested for enrichment relative to a background setusing the hypergeometric distribution. miRNA target regulation wasmeasured by enrichment of transcripts containing miRNA hexamer seedstrings (stretches of 6 contiguous bases complementary to miRNA seedregion nucleotide positions 1-6, 2-7, or 3-8) in transcripts havingannotated 3′UTRs.

TABLE 5 Expression alterations for miR-34 down-regulated genes in HCT116Dicer Ex5 cells. Genbank Luc Gene Names Accession #^(b) siRNA mir-34amir-34b mir-34c TK1 NM_003258 1.09 3.65 3.22 3.02 PHF19 AL117477 1.013.31 2.40 2.99 MET AK025784 1.64 3.24 3.16 2.88 LOC149832 BC044234 1.293.06 2.61 2.42 MCM3 NM_002388 1.21 3.03 2.86 3.27 FLJ11029 AW183918−1.14 2.97 2.02 1.99 SH3GL1 NM_003025 1.47 2.97 2.44 2.25 FGFRL1NM_021923 1.08 2.94 3.04 3.09 CHES1 NM_005197 1.34 2.79 2.58 2.50PPP1R11 NM_021959 1.10 2.75 2.31 2.29 MGC5508 NM_024092 1.25 2.75 2.912.86 CDK4 NM_052984 1.07 2.74 2.39 2.29 C1orf19 NM_052965 −1.01 2.722.50 2.57 NUP210 NM_024923 1.01 2.71 1.86 1.75 RAB21 BC009109 −1.03 2.701.89 1.57 SLC35A4 NM_080670 1.08 2.70 1.99 1.87 NASP NM_172164 1.19 2.682.56 2.73 ANKRD40 AK054795 1.04 2.68 2.04 2.20 MGC5242 AK056910 −1.112.67 2.34 2.33 SGPP1 AI762918 −1.01 2.64 1.87 1.84 LMAN2L NM_030805−1.06 2.64 2.54 2.60 ULBP2 NM_025217 1.42 2.62 2.54 3.09 FKSG24NM_032683 1.04 2.61 2.00 2.00 CNOT6 NM_015455 −1.10 2.59 1.68 1.66 CAP1NM_006367 −1.00 2.59 1.86 1.63 MGC16207 BC007379 1.18 2.57 2.54 2.47FLJ11029 NM_018304 −1.22 2.57 1.93 2.02 E2F2 AF086395 1.31 2.57 1.932.20 TPD52 NM_005079 1.02 2.56 1.82 1.85 TTC19 NM_017775 1.03 2.56 2.052.24 GLRX5 NM_016417 −1.04 2.50 1.98 1.81 MYB NM_005375 1.32 2.50 1.902.17 ATG9A NM_024085 1.04 2.48 1.81 1.93 VAMP2 NM_014232 1.02 2.45 1.972.52 SLC29A1 NM_004955 1.24 2.45 1.71 1.80 FAM64A NM_019013 −1.11 2.441.97 1.96 CDCA5 NM_080668 1.15 2.44 2.12 2.29 CDC25A AI343459 1.03 2.402.04 2.20 FURIN NM_002569 −1.08 2.39 1.74 1.68 DTL NM_016448 1.23 2.392.82 3.32 TMED8 AK095650 −1.08 2.38 2.63 2.50 SHCBP1 NM_024745 −1.052.38 2.72 2.96 TRIB3 NM_021158 1.08 2.37 1.86 1.88 MET NM_000245 1.292.36 2.57 2.27 RKHD2 NM_016626 −1.05 2.36 2.47 2.43 GMNN NM_015895 −1.082.35 2.08 2.09 ARHGAP1 NM_004308 1.71 2.34 1.96 1.99 PKMYT1 NM_0042031.16 2.31 1.73 1.99 MGC13170 NM_032712 1.15 2.31 1.56 1.65 C6orf89AJ420511 1.06 2.31 2.53 2.29 TSPAN14 NM_030927 1.10 2.30 1.54 1.37FLJ13912 NM_022770 1.05 2.25 2.19 2.35 CDK6 AI333092 1.08 2.25 2.36 1.80MAP3K11 NM_002419 −1.06 2.23 1.78 1.82 CTDSPL NM_005808 1.21 2.23 2.242.74 CDS2 AI972315 1.05 2.22 1.89 1.92 SLC44A2 NM_020428 1.05 2.22 2.081.97 TGIF2 NM_021809 1.05 2.22 2.30 2.43 MYOHD1 NM_025109 1.00 2.21 1.611.83 CTDSP2 NM_005730 −1.03 2.21 1.66 −2.00 SURF4 NM_033161 1.26 2.191.93 1.82 YKT6 NM_006555 1.02 2.19 1.71 1.62 CDC23 NM_004661 1.14 2.191.76 1.76 GNPDA1 NM_005471 1.45 2.18 1.71 1.48 NAGPA NM_016256 1.03 2.171.85 2.07 RDH11 NM_016026 1.11 2.14 1.73 1.61 IMPDH1 NM_000883 1.23 2.131.77 1.67 SPBC25 NM_020675 −1.12 2.12 2.09 1.94 SPFH1 NM_006459 −1.002.11 2.48 2.54 PHGDH NM_006623 1.21 2.10 2.38 2.17 CHES1 NM_018589 1.132.09 2.18 2.22 CCNE2 NM_057749 1.42 2.08 2.08 2.34 XBP1 NM_005080 1.182.07 2.04 1.99 RAD54L NM_003579 1.07 2.06 1.85 2.29 RDX NM_002906 −1.032.05 1.75 1.95 FLJ14154 NM_024845 1.68 2.04 2.01 2.01 SIX5 NM_1758751.07 2.03 1.82 1.98 FANCA NM_000135 1.11 2.03 1.70 2.22 KIAA1333NM_017769 1.13 2.03 1.59 1.66 C8orf55 NM_016647 −1.07 2.03 1.83 1.74MGC21644 NM_182960 −1.30 2.02 1.75 2.20 TMEM48 NM_018087 −1.06 2.02 1.811.74 FANCG NM_004629 −1.02 2.01 1.61 1.84 CPSF6 NM_007007 1.04 2.01 2.082.45 CCNE2 NM_004702 1.26 2.01 2.08 2.39 MCM5 NM_006739 1.09 2.01 1.561.69 CTDSP1 NM_021198 −1.00 2.00 1.69 1.86 DKFZp564K142 NM_032121 −1.262.00 1.96 2.31 AXL NM_001699 1.03 1.99 1.61 1.87 KIAA0101 NM_014736−1.19 1.99 1.59 1.56 STMN1 NM_005563 −1.04 1.98 2.13 2.11 TAF5 NM_139052−1.07 1.98 2.01 2.11 MBD3 AL390153 1.08 1.97 1.86 1.89 FBXO10 BC013747−1.24 1.97 1.43 1.69 C7orf21 NM_031434 −1.14 1.95 1.73 1.93 HMMRNM_012484 −1.22 1.95 2.28 2.44 UBE2L3 NM_003347 1.26 1.95 1.69 1.54SGPP1 NM_030791 −1.20 1.94 1.68 1.53 MYBL2 NM_002466 1.03 1.94 1.87 2.08RPAP1 NM_015540 1.20 1.93 1.95 2.03 MGC5242 NM_024033 −1.12 1.93 1.982.11 LASS2 NM_022075 1.27 1.92 1.78 1.92 VPS4A NM_013245 −1.05 1.92 1.921.95 ZDHHC16 NM_032327 −1.05 1.92 1.62 1.46 LRRC40 NM_017768 −1.16 1.921.82 1.86 C9orf140 NM_178448 −1.01 1.91 1.64 1.61 WDR76 AI220472 1.101.91 1.83 2.08 MGC23280 NM_144683 1.08 1.91 1.45 1.52 UNC84B NM_0153741.02 1.91 1.69 1.56 VCL NM_003373 −1.13 1.90 1.61 1.68 SNX15 NM_0133061.05 1.89 1.73 1.80 ARAF NM_001654 1.15 1.89 1.58 1.59 C20orf100NM_032883 1.04 1.89 1.54 1.60 CUEDC1 AI936146 1.23 1.89 1.69 1.90 BRCA1NM_007300 1.02 1.88 2.31 2.67 SFRS1 AI589112 1.10 1.88 1.53 1.73 TSNNM_004622 −1.12 1.87 1.88 1.71 CUEDC1 NM_017949 1.18 1.85 1.66 1.78GAS2L3 NM_174942 −1.01 1.85 1.39 1.41 ZNF358 NM_018083 1.02 1.84 1.661.56 HTLF AA827684 1.20 1.84 2.12 2.01 SCRIB NM_015356 1.06 1.83 1.631.81 DKFZP564O0823 AK025205 1.17 1.83 3.17 3.23 GSG2 NM_031965 −1.121.83 2.08 2.27 WDR62 NM_015671 1.04 1.82 1.59 1.95 GOLPH3L NM_018178−1.03 1.82 2.18 2.14 PER2 NM_022817 −1.04 1.82 1.28 1.19 FEN1 NM_004111−1.11 1.81 2.05 2.20 ERO1L AK024224 1.36 1.81 1.83 1.94 CD151 NM_0043571.13 1.81 1.65 1.58 C6orf89 AK001957 −1.09 1.81 2.05 2.08 ZNF395NM_017606 1.00 1.80 2.26 2.02 HMGN4 NM_006353 −1.08 1.80 2.92 2.88 EME1NM_152463 −1.05 1.79 1.79 2.26 RP13-15M17.2 AI953008 1.08 1.79 1.88 1.80CIC NM_015125 1.05 1.79 1.47 1.53 MBD3 NM_003926 1.15 1.78 1.36 1.46KIAA1704 AB051491 −1.06 1.78 1.30 1.41 AXL NM_021913 1.00 1.78 1.59 1.75PSF1 D80008 −1.16 1.78 1.81 1.99 BRRN1 NM_015341 1.04 1.78 1.69 1.84SLC45A3 NM_033102 1.25 1.77 1.55 1.73 CASKIN2 NM_020753 1.14 1.77 1.551.60 CHAF1A NM_005483 1.13 1.77 1.65 1.96 RASSF5 NM_031437 1.07 1.771.83 2.05 F8 NM_019863 −1.13 1.77 1.57 1.42 MGC12538 AA703254 1.39 1.761.56 1.24 C9orf125 AJ420439 1.02 1.76 2.10 2.09 RAD51 NM_002875 1.161.76 1.58 1.77 HDAC1 NM_004964 −1.07 1.76 1.96 1.96 NFYC NM_014223 −1.041.76 1.73 1.97 HIST1H4E NM_003545 1.07 1.75 1.66 1.83 PLK1 NM_0050301.15 1.75 1.61 1.68 PTP4A2 NM_080391 1.23 1.74 2.29 2.60 LOC159090AL832218 −1.08 1.74 1.86 2.00 TOM1L2 AL133641 1.01 1.74 1.45 1.42 FEM1ANM_018708 1.00 1.74 1.42 1.26 TESK1 NM_006285 −1.03 1.74 1.67 1.87UBE2Q1 NM_017582 1.18 1.74 2.24 2.38 ESPL1 NM_012291 −1.03 1.74 1.561.61 RRM2 BC028932 1.05 1.74 2.05 2.32 SCMH1 NM_012236 1.10 1.74 1.762.04 SFXN5 NM_144579 1.02 1.73 1.76 1.90 MTA2 NM_004739 1.19 1.73 1.561.54 SURF5 NM_006752 1.04 1.73 1.47 1.64 SLC16A4 AK091279 1.04 1.73 1.491.69 FUT8 NM_004480 −1.03 1.73 1.76 1.75 DTYMK NM_012145 −1.01 1.72 1.351.43 ATP1B3 NM_001679 1.00 1.72 1.77 1.64 SPBC24 NM_182513 −1.09 1.721.46 1.68 FLJ37034 BC047423 −1.01 1.72 1.79 1.98 FLJ13868 NM_022744 1.021.72 1.48 1.47 BCL2 NM_000633 −1.03 1.72 1.46 1.44 CKLF AI077541 −1.081.72 1.49 1.58 C10orf38 AL050367 1.05 1.71 1.45 1.46 CABLES2 BC003122−1.16 1.71 1.61 1.69 FLJ39827 NM_152424 1.38 1.71 1.47 1.39 MDM4NM_002393 −1.16 1.71 1.34 1.44 FAM100B NM_182565 −1.10 1.71 1.64 1.69ZDHHC12 NM_032799 1.05 1.71 1.50 1.40 KIAA1160 NM_020701 −1.12 1.71 1.451.49 ACSL4 NM_022977 −1.01 1.71 2.06 2.04 ZHX2 NM_014943 1.09 1.71 1.701.60 KIF11 NM_004523 −1.04 1.71 1.59 1.69 GTSE1 NM_016426 1.02 1.70 1.631.76 DDX10 NM_004398 1.18 1.70 1.49 1.35 NQO1 NM_000903 0.00 1.70 2.932.27 ORC1L NM_004153 1.11 1.70 1.91 2.32 PURB AK057669 1.08 1.70 1.791.80 FLJ14166 NM_024565 −1.10 1.69 1.77 1.90 TBC1D13 NM_018201 1.15 1.691.49 1.86 PMF1 NM_007221 1.05 1.69 1.75 1.69 IFRD2 NM_006764 1.02 1.691.87 2.01 AFG3L1 NM_001132 −1.19 1.68 1.63 2.19 CEP55 NM_018131 −1.221.68 1.48 1.52 MKI67 NM_002417 −1.16 1.68 1.58 1.40 PLAGL2 NM_0026571.04 1.68 1.50 1.67 VCL NM_014000 −1.18 1.68 1.48 1.58 ARHGDIB NM_001175−1.11 1.68 1.58 1.87 UBE2C NM_181802 −1.03 1.68 1.43 1.50 KCNS3NM_002252 −1.08 1.68 1.72 1.59 CCDC15 NM_025004 −1.03 1.67 1.46 1.60LASS5 NM_147190 −1.03 1.67 1.66 1.63 PALLD NM_016081 1.02 1.67 1.41 1.28AREG NM_001657 1.56 1.67 1.62 1.35 PTTG3 NM_021000 0.00 1.66 1.47 1.51BIRC5 NM_001168 −1.18 1.66 1.89 1.98 UBE2C NM_007019 −1.05 1.66 1.421.52 ABR NM_001092 1.25 1.66 1.39 1.56 ZNF580 NM_016202 1.05 1.66 1.601.54 PHF17 NM_024900 1.02 1.65 1.48 1.49 NMT1 NM_021079 1.03 1.65 2.442.58 PHB NM_002634 1.08 1.65 1.45 1.44 Pfs2 NM_016095 1.16 1.65 1.531.74 NDP52 NM_005831 −1.01 1.65 1.42 1.30 DKFZp762E1312 NM_018410 1.031.65 1.51 1.78 C9orf10OS AK056096 −1.06 1.64 1.45 1.62 DDX11 NM_004399−1.01 1.64 1.53 2.14 GCH1 NM_000161 1.35 1.64 1.70 1.61 RNF38 NM_022781−1.08 1.64 1.47 1.32 FSHPRH1 AI190209 −1.01 1.64 1.75 2.07 LOC388730AI420422 1.14 1.64 1.39 1.37 PARP16 NM_017851 1.10 1.64 2.04 2.20 MAPK9AI096774 1.03 1.64 1.54 1.53 C14orf94 NM_017815 1.02 1.63 1.41 1.50 MPP2NM_005374 1.07 1.63 1.73 1.43 FAM49B AA497060 1.35 1.63 1.83 1.84 HPCAL4NM_016257 −1.07 1.63 1.96 2.02 WHSC1 NM_133336 1.50 1.63 1.99 2.25C15orf21 NM_173609 1.07 1.63 1.53 1.53 MFN2 NM_014874 1.03 1.63 1.501.29 LOC146517 AL833385 −1.04 1.62 1.45 1.62 ORC6L NM_014321 1.17 1.621.51 1.71 QDPR NM_000320 −1.00 1.62 1.72 1.70 POLQ NM_006596 −1.01 1.621.46 1.67 KIF15 NM_020242 −1.00 1.62 1.92 2.13 GRPEL2 NM_152407 1.041.62 1.89 1.98 FLJ20255 NM_017728 1.13 1.62 1.46 1.61 ZNF395 NM_0186601.03 1.61 1.81 1.69 HMGB3 NM_005342 −1.03 1.61 1.77 1.88 UBP1 NM_0145171.06 1.61 2.08 2.20 WHSC1 NM_133330 1.32 1.61 2.10 2.28 TATDN2 NM_0147601.06 1.61 1.77 1.83 HIRIP3 NM_003609 1.11 1.61 1.39 1.44 ZNF551NM_138347 1.00 1.60 1.33 1.51 TUBA2 NM_006001 1.04 1.60 1.39 1.31 ATPAF1AL137294 −1.20 1.60 1.59 1.39 RANBP10 AB040897 −1.02 1.60 1.57 1.75MAC30 NM_014573 1.06 1.59 1.42 1.44 HIP2 AL117568 −1.05 1.59 2.11 2.06CAV1 AF074993 1.23 1.59 1.52 1.60 EXOSC2 NM_014285 1.19 1.59 1.51 1.65ASXL1 NM_015338 1.01 1.59 1.60 1.77 AI890133 −1.07 1.59 1.48 1.29KIAA1160 AK024035 1.05 1.59 1.28 1.39 TUBAP NG_000900 1.08 1.59 1.351.36 MED8 NM_052877 1.01 1.59 1.80 1.91 CDK6 AK000660 −1.26 1.58 1.991.85 KIFC1 NM_002263 −1.01 1.58 1.56 1.96 RP13-360B22.2 NM_032227 1.021.58 1.73 1.80 EXO1 NM_130398 1.07 1.58 1.45 1.65 EFNA5 AW015347 1.111.58 1.85 1.83 CCND3 NM_001760 −1.13 1.58 1.68 1.83 MAP2K1 NM_002755−1.14 1.57 1.96 2.23 FAM76A AI805069 −1.10 1.57 1.39 1.57 C9orf25NM_147202 −1.17 1.57 1.48 1.69 W93501 −1.13 1.56 1.56 1.65 BARD1NM_000465 1.15 1.56 1.42 1.83 ADRBK2 BC029563 −1.05 1.56 1.59 1.50CDC25C NM_001790 1.01 1.56 1.37 1.40 FLJ20232 NM_019008 1.03 1.56 1.881.84 POU2F1 BC037864 1.15 1.56 1.93 1.64 NDRG1 NM_006096 1.29 1.56 2.032.03 PSMB7 AJ420421 1.04 1.56 1.34 1.32 D4ST1 NM_130468 1.02 1.56 1.791.85 CCNF NM_001761 1.01 1.56 1.61 1.76 CDKN3 NM_005192 −1.34 1.56 1.401.30 PRR3 NM_025263 −1.20 1.55 1.39 1.48 FADS2 NM_004265 1.11 1.55 1.511.62 FANCE NM_021922 1.03 1.55 1.25 1.37 CAV1 NM_001753 1.26 1.55 1.451.34 SAMD6 NM_173551 1.05 1.54 1.55 1.59 BID AK057062 1.03 1.54 1.591.62 FIGNL1 NM_022116 −1.04 1.54 1.23 1.28 CENPF NM_016343 1.00 1.541.62 1.65 DKFZp586I1420 NM_152747 1.05 1.54 1.38 1.37 E2F8 NM_0246801.04 1.54 1.55 1.90 SLC7A1 AL050021 1.16 1.54 1.70 1.51 HCN3 AB0409681.09 1.54 1.32 2.07 KIF20A NM_005733 1.03 1.54 1.41 1.50 DGKZ NM_0036461.11 1.54 1.52 1.67 DCLRE1B NM_022836 −1.01 1.54 1.52 1.84 DHCR24NM_014762 1.16 1.53 1.42 1.52 ETEA NM_014613 1.23 1.53 1.28 1.28 PHF6NM_032458 −1.03 1.53 2.25 2.21 CDC45L NM_003504 1.04 1.53 1.80 2.21C8orf30A NM_016458 1.03 1.53 1.74 1.75 HMGB3 BC007608 1.05 1.53 1.922.03 RARG NM_000966 1.02 1.53 1.55 1.47 NUSAP1 NM_016359 1.03 1.53 1.451.50 ASF1B NM_018154 −1.04 1.53 1.60 1.76 MMS19L NM_022362 1.09 1.531.47 1.55 ACSL4 NM_004458 −1.09 1.53 1.95 1.91 TRAF7 NM_032271 1.26 1.531.33 1.36 C15orf42 NM_152259 1.08 1.53 1.43 1.62 CDCA8 NM_018101 1.041.52 1.62 1.72 UHRF2 NM_152306 1.07 1.52 1.26 1.43 FOXM1 NM_021953 −1.131.52 1.38 1.52 C22orf18 NM_024053 1.10 1.52 1.53 1.57 EVI5L NM_145245−1.02 1.52 1.70 1.69 AADACL1 NM_020792 1.33 1.52 1.73 1.82 ATP1B3P1NG_000849 −1.13 1.51 1.62 1.52 TRIOBP NM_138632 1.24 1.51 1.46 1.52 FUT8NM_178155 1.00 1.51 1.48 1.53 IQGAP3 NM_178229 1.08 1.51 1.23 1.43METTL1 NM_005371 1.11 1.51 1.55 1.57 OATL1 L08240 1.06 1.51 1.35 1.30WSB2 NM_018639 1.33 1.50 1.36 1.32 ETV5 NM_004454 −1.07 1.50 1.67 1.83C21orf63 NM_058187 −1.19 1.50 1.25 1.28 ENST00000273097 ENST00000273097−1.01 1.50 1.53 1.45 TBPIP NM_013290 −1.15 1.50 1.53 1.80 VDR NM_0003761.00 1.50 1.50 1.51 FKBP1B NM_054033 1.24 1.50 1.68 1.64 CSRP1 NM_0040781.14 1.50 1.65 1.85 RRAS NM_006270 −1.02 1.50 1.38 1.42 BTRC NM_032715−1.04 1.50 1.28 1.37 IRAK1 NM_001569 1.07 1.50 1.53 1.60 MTMR9 NM_015458−1.04 1.49 1.73 1.79 FBXO5 NM_012177 −1.12 1.49 1.46 1.65 MGAT2NM_002408 1.11 1.49 1.37 1.42 CHMP7 NM_152272 −1.00 1.49 1.43 1.41R3HDM1 NM_015361 1.01 1.49 1.44 1.45 FLJ32363 BC036867 −1.14 1.49 1.541.70 Ells1 NM_152793 1.17 1.49 1.93 2.05 MGC13024 NM_152288 −1.10 1.491.23 1.19 FOXJ2 NM_018416 1.11 1.49 1.29 1.25 PBEF1 NM_005746 1.02 1.481.51 1.37 H2AFX NM_002105 −1.03 1.48 1.53 1.54 TESK2 NM_007170 1.01 1.481.51 1.89 OXSR1 NM_005109 −1.06 1.48 1.58 1.50 RAD51C NM_002876 1.021.48 1.34 1.33 RIC8B NM_018157 −1.13 1.48 1.40 1.48 KLHDC3 NM_057161−1.11 1.48 1.89 2.25 RBM12 NM_152838 1.12 1.48 1.33 1.24 DGAT1 NM_0120791.17 1.48 1.39 1.62 STX1A NM_004603 1.09 1.48 1.18 1.55 GSK3B AW1395381.14 1.48 1.65 1.59 MKL1 NM_020831 1.02 1.47 1.41 1.57 LASS2 NM_0133841.11 1.47 1.57 1.56 MLF1IP NM_024629 1.03 1.47 1.50 1.65 SCNN1ANM_001038 −1.05 1.47 1.34 1.27 PRC1 NM_003981 1.04 1.47 1.53 1.64 USP3AK094444 1.23 1.47 1.94 1.86 FLJ39660 NM_173466 −1.08 1.47 1.61 2.20PPARG NM_005037 −1.06 1.47 1.82 1.86 EIF2AK1 NM_014413 1.38 1.47 2.222.09 TMEM22 NM_025246 −1.07 1.47 1.53 1.54 HSPC142 NM_014173 −1.03 1.471.31 1.30 C10orf26 AK000161 −1.28 1.47 1.53 1.41 C6orf106 NM_022758 1.031.47 1.54 1.70 SMPD1 NM_000543 −1.17 1.47 1.34 1.33 RRM1 NM_001033 1.101.46 1.31 1.38 MSH6 NM_000179 1.01 1.46 1.49 1.63 PPIG R38692 1.05 1.461.46 1.43 KIF22 NM_007317 1.02 1.46 1.38 1.47 USP15 NM_006313 1.08 1.461.58 1.56 LOC400927 AW206718 1.10 1.46 1.37 1.36 PTTG1 NM_004219 −1.061.46 1.32 1.40 PPM1A BM676083 −1.04 1.46 1.70 1.48 ST3GAL5 NM_0038961.49 1.46 1.68 1.66 CENPJ NM_018451 1.05 1.46 1.48 1.82 S100A2 NM_005978−1.02 1.46 1.49 1.39 PPRC1 NM_015062 1.11 1.46 1.27 1.53 LOC441347AL050136 −1.11 1.46 1.80 1.55 FLOT2 NM_004475 −1.02 1.46 1.74 1.69 CDC7NM_003503 1.02 1.45 1.48 1.72 KIAA0157 NM_032182 1.01 1.45 1.88 1.96AK024294 1.14 1.45 1.52 1.35 FUT8 NM_178154 1.03 1.45 1.52 1.49 SENP1BC045639 −1.05 1.45 1.62 1.71 TNFRSF1A NM_001065 1.06 1.45 1.31 1.36ARSB AK026942 −1.04 1.45 1.58 1.61 TTK NM_003318 −1.08 1.45 1.35 1.41KIAA0984 AB023201 1.01 1.44 1.93 1.98 RFC4 NM_181573 1.00 1.44 1.59 1.78CLSPN NM_022111 −1.10 1.44 1.48 1.52 AOC3 NM_003734 −1.05 1.44 1.22 1.50PSRC1 NM_032636 −1.10 1.44 1.46 1.65 CREB3L2 AL080209 1.02 1.44 1.941.71 TPT1 AI803535 1.04 1.44 1.41 1.39 MAP3K7IP2 NM_145342 −1.13 1.441.56 1.52 C18orf24 NM_145060 −1.03 1.44 2.10 2.44 STK39 NM_013233 1.101.44 1.17 1.04 KIAA0476 NM_014856 1.02 1.43 1.31 1.60 GRK6 NM_0020821.06 1.43 1.58 1.41 FARP1 AK025683 1.01 1.43 1.42 1.25 FLJ22794NM_022074 1.07 1.43 1.49 1.80 MGC18216 NM_152452 1.76 1.43 1.27 1.08WHSC1 NM_133334 1.05 1.43 1.72 1.92 TROAP NM_005480 −1.01 1.43 1.40 1.69PRIM1 NM_000946 1.16 1.43 1.46 1.44 TMEM55A NM_018710 −1.11 1.43 1.431.46 LSS NM_002340 1.17 1.42 1.31 1.36 PURB AK056651 −1.16 1.42 1.642.26 LOC151162 AF055029 1.27 1.42 2.23 2.24 BLM NM_000057 1.16 1.42 1.701.97 LONRF2 AL157505 −1.17 1.42 1.43 1.36 AI927895 1.06 1.42 1.80 1.87KLC2 NM_022822 1.00 1.42 1.36 1.45 STCH NM_006948 −1.07 1.42 1.54 1.51PTTG2 NM_006607 −1.09 1.42 1.33 1.38 GDPD5 NM_030792 −1.11 1.42 1.351.47 CRTC2 NM_181715 1.08 1.42 1.33 1.47 DCTN5 NM_032486 1.24 1.42 1.571.73 POU2F1 NM_002697 1.04 1.42 1.45 1.33 KIF4A NM_012310 −1.02 1.421.30 1.40 ESAM NM_138961 −1.12 1.42 1.28 1.43 JPH1 NM_020647 −1.08 1.421.49 1.40 OVOS2 NM_173498 −1.08 1.41 1.28 1.37 ATF4 NM_001675 −1.00 1.411.38 1.32 CKLF NM_016951 1.02 1.41 1.37 1.48 NT5E AA046478 1.09 1.411.46 1.65 SLC12A2 AK025062 1.23 1.41 1.59 1.76 hCAP-D3 D29954 −1.12 1.411.39 1.38 LMNB1 NM_005573 1.05 1.41 1.57 1.33 ATG5 NM_004849 1.30 1.411.98 1.95 SEMA4F NM_004263 1.03 1.41 1.28 1.29 ZDHHC8 NM_013373 1.101.40 1.14 1.52 NXF4 ENST00000289078 1.05 1.40 1.43 1.59 HCAP-G NM_0223461.06 1.40 1.96 2.18 PNPLA2 NM_020376 1.14 1.40 1.39 1.46 FAM76ANM_152660 −1.03 1.40 1.39 1.40 RDH5 NM_002905 1.04 1.40 1.42 1.55 FSBPNM_006550 −1.06 1.40 1.37 1.55 XPO4 NM_022459 1.05 1.40 1.09 1.16 MTMR10AL833089 −1.02 1.40 1.67 1.75 C21orf59 AI564020 1.08 1.40 1.50 1.58C15orf20 AF108138 −1.09 1.40 1.59 2.25 TBPIP NM_016556 −1.23 1.40 1.481.73 L3MBTL3 AB058701 1.01 1.39 1.41 1.30 TUBA3 NM_006009 −1.07 1.391.46 1.28 XRCC3 NM_005432 1.13 1.39 1.38 1.63 TFCP2L1 NM_014553 1.261.39 1.61 2.15 MCM10 NM_018518 1.21 1.39 1.63 1.96 FLJ38608 NM_153215−1.03 1.39 1.49 1.42 FLJ13710 AI608673 1.07 1.39 1.26 1.15 GGA2NM_015044 1.23 1.39 1.27 1.41 FAM62B AB033054 1.03 1.39 1.49 1.51 FUT1NM_000148 1.01 1.39 1.24 1.31 DHX33 AA534526 1.01 1.38 1.47 1.52 TRIM6NM_058166 −1.52 1.38 1.74 1.99 PPP2R3B NM_013239 −1.07 1.38 1.22 1.82TNPO1 AL049378 1.33 1.38 1.52 1.61 C6orf153 NM_033112 1.03 1.38 1.331.42 C2orf7 NM_032319 −1.05 1.38 1.34 1.61 HNRPR AK001846 1.11 1.38 1.731.76 PRKAA1 AI375852 −1.18 1.38 1.37 1.57 SLC19A1 NM_003056 1.12 1.381.35 1.45 C17orf41 NM_024857 −1.16 1.38 1.45 1.65 EZH2 NM_152998 1.121.38 1.58 1.83 C10orf119 NM_024834 1.27 1.38 1.78 1.86 AK021744 AK0217441.18 1.37 1.17 −1.15 DHX37 NM_032656 1.17 1.37 1.30 1.38 MECP2 NM_0049921.29 1.37 1.74 1.70 LGALS1 NM_002305 −1.01 1.37 0.00 0.00 CCNB2NM_004701 −1.04 1.37 1.54 1.60 LOC388134 AL355708 −1.01 1.37 1.40 1.09LYPLAL1 NM_138794 −1.12 1.37 1.40 1.15 SRGAP2 AB007925 −1.10 1.37 1.501.44 ARHGEF5 NM_005435 1.11 1.37 1.27 1.36 SHMT1 NM_004169 −1.04 1.361.42 1.45 DDR1 NM_001954 1.10 1.36 1.38 1.48 TACC3 NM_006342 −1.00 1.361.34 1.50 FLJ27365 AI973033 1.01 1.36 1.30 1.57 ECOP NM_030796 1.08 1.361.35 1.63 PTTG1IP NM_004339 1.12 1.36 1.42 1.45 RRM2 NM_001034 −1.111.36 1.89 1.91 DHX33 NM_020162 −1.06 1.36 1.38 1.47 PSD3 NM_018422 1.041.35 1.17 −1.08 COPS7B NM_022730 1.28 1.35 1.45 1.55 CDCA1 NM_031423−1.20 1.35 1.51 1.70 ^(a)Each value represents fold reduction for eachexperimental condition as indicated, as compared to the mocktransfection in Hct116 Dicer^(ex5) cells. Negative value in the lucsiRNA transfected cells represents fold increase. ^(b)Refseq accessionnumbers are provided for all annotated genes, which are each herebyincorporated by reference. mRNA accession numbers are provided for thoseunannotated genes included on the microarray.

Consistent with the cell cycle phenotype described in Example 2, thegenes listed in Table 5 were found to be enriched for genes associatedwith cell cycle (see Table 6). In addition, the down-regulated orup-regulated gene signatures of the microarray data were examined todetermine whether genes associated with TP53 pathway were enriched ineither of these miR-34 response gene signatures. To do this, the datawere examined to determine the degree of overlap between the miR-34signature genes and TP53 pathway gene identified as such in the GeneOntology Database (Camon et al., 2004, Nucleic Acids Res. 32:D262-6;Camon et al., 2003, Genome Res. 13:662-72), the TP53 DNA damage responsegene set, genes identified as being down-regulated in the RNAiexperiments reported herein, and a set of direct TP53 targets identifiedby a genome-scale chromatin immunoprecipitation (ChIP) analysis of TP53transcription factor binding sites (Wei et al., 2006, Cell 124:207-19).

Table 6 provides the overlap of genes up-regulated or down-regulated bymiR-34 transfection with sets of genes implicated in DNA damage and thecell cycle. Biological function was categorized by enrichment oftranscripts from Gene Ontology Biological Process functional categories(http://www.geneontology.org/), as described in The Gene OntologyConsortium, Gene Ontology: tool for the unification of biology. NatureGenetics (2000) 25:25-29. The numbers of genes in the identified genesets or the overlaps of the sets are shown in brackets and italicizedfont. The probability of each result, expressed as a P-value, wascalculated by hypergeometric distribution (Lee et al., BMCBioinformatics 2005 6:189) and is shown in Table 6. All genesrepresented on the microarray were used as the background set.

TABLE 6 Overlap of genes up-regulated or down-regulated by miR-34transfection with sets of genes implicated in DNA damage and the cellcycle. Genes down-regulated Genes up-regulated GO Biological ProcessGene Set Categories following doxorubicin following doxorubicin categoryfor “Cell Being Compared treatment [2104 genes] treatment [2280 genes]cycle” [1202 genes] Genes up-regulated by P-value: 0.56 P-value: 7.0e−65P-value: 7.4e−5 miR-34 [1022 genes] [Overlap: 101 genes] [Overlap: 303genes] [Overlap: 88 genes] Genes down-regulated by P-value: 1.8e−73P-value: 0.94 P-value: 1.1e−19 miR-34 [582 genes] [Overlap: 219 genes][Overlap: 52 genes] [Overlap: 93 genes]

A significant overlap between miR-34-regulated genes and those whoseexpression is altered upon DNA damage (Table 6) was observed. In thiscase, significant overlap was seen both for genes that increased inresponse to miR-34 transfection (p<7e-65) and those that are repressedupon miR-34 activation (p<1.8e-73). However, while a strong enrichmentof genes that have sequences complementary to miR-34 seed regions wasseen in the down-regulated overlapping set, it was not seen in theup-regulated gene set, suggesting that the genes up-regulated inexpression might be caused by secondary effects of miR-34.

As shown in Table 6, a significant overlap was found between genesregulated by miR34a and common TP53 mediated events, suggesting thatmiR-34 transfection may induce at least a portion of the TP53 pathway.

Example 4

This Example demonstrates that introduction of synthetic miR449, amember of the miR-34 family, into cell line HCT116 elicits a phenotypesimilar to that induced by activation of the TP53 G1 checkpoint.

Rationale:

Delay of the G1/S transition of the cell cycle is known to be aconsequence of TP 53 activation. In this example, miR-449 siRNA duplexeswere designed with passenger strands that are complementary to thenatural mature miRNA, except for a single base mismatch four bases fromthe 3′ end of the sequence, referred to as “asymmetric passengerstrands.” Exemplary asymmetric passenger strands are provided in Table 7for miR-449 (SEQ ID NO:32), with the mismatch underlined. As shown inTable 7, the synthetically designed asymmetric passenger strand formiR-449 (SEQ ID NO:32) differs from the natural passenger strand formiR-449 (SEQ ID NO:38). The data presented in this example shows thatintroduction of duplex a miR-449 mimetic comprising a natural miR449guide strand (SEQ ID NO:29) annealed to a asymmetric miR-449 passengerstrand (SEQ ID NO:32) into cells leads to cell cycle arrest at the G1checkpoint in a manner that is analogous to activation of TP53.

Methods:

The cell line HCT116#2, a p53 positive cell line, was transfected withmiR-16, miR-34a, miR-34a-mm-2,3, miR-449, and miR-449-mm-2,3 orluciferase control using the DNA oligonucleotides described in Table 7.Prior to transfection, the cells were seeded at 12.5×104 and transfectedusing 10 nM final concentration of the synthetic oligonucleotides usingLipofectamine RNAiMax. 30 hours post transfection, Nocodazole was addedat a final concentration of 100 ng/mL. The cells were harvested 18 hoursafter adding nocodazole.

TABLE 7 Synthetic miR-449 Oligonucleotide Sequences siRNA, miRNA or SEQSEQ mismatch Guide strand/mature ID Passenger strand ID miRNA (5′ to 3′)NO: (5′ to 3′) NO: miR34a UGGCAGUGUCUUAGCUGGUUGU 1AACCAGCUAAGACACUGCGAAU 12 (natural) (synthetic: reverse complement ofnatural guide strand with one base mismatch) miR34a-UCCCAGUGUCUUAGCUGGUUGU 13 AACCAGCUAAGACACUGGCAAU 14 mm2,3(mutation in seed region) (synthetic: reverse complement ofseed region mutation with one base mismatch) miR449UGGCAGUGUAUUGUUAGCUGGU 29 AUCGGCUAACAUGCAACUGCUG 38 (natural) (natural)miR449 UGGCAGUGUAUUGUUAGCUGGU 29 CAGCUAACAAUACACUGUUAAU 32 (natural)(synthetic: reverse complement of natural guide strand with mismatch)miR449_ UCCCAGUGUAUUGUUAGCUGGU 33 CAGCUAACAAUACACUGGCAAU 34 mm2,3(mutation in seed region) (synthetic: reverse complement of (seedseed region mutation) mismatch) luciferase CGUACGCGGAAUACUUCGA 27UCGAAGUAUUCCGUACG 28

As shown in Table 8, the transfection of miR-449 (WT mature) and miR-34a(WT mature) results in a G1 arrest of HCT116 cells, similar to theresults observed when miR-34a (WT mature), miR-34b (WT mature), andmiR-34c (WT mature) were transfected into A549 cells, as shown inExample 2. As further shown in Table 8, transfection of miR-16 (WTmature) also results in a G1 arrest of HCT116 cells, consistent with theresults described in Linsley P. S. et al., Mol Cell Biol 27: 2240-2252(2007).

TABLE 8 Cell Cycle Arrest in HCT116 Cells (wild type p53) Transfectedwith Synthetic siRNA Constructs microRNA species introduced into % CellsHCT116 cells (wt p53) in G1 miR34a (WT mature) 52.9% miR34a-mm2,3 (seedmismatch) 8.8% miR449 (WT mature) 40.9% miR449 (seed mismatch) 9.2%luciferase 9.5% miR16 (WT mature: positive control) 60.5% mocktransfection 8.2%

Discussion: miR-34s belong to an evolutionary conserved miRNA family,with single, recognizable orthologues in several invertebrate species.See He et al., Nature 447:1130-1134 (2007). As shown in FIG. 1, the seedregion of miR-449 (SEQ ID NO:31) comprises a nucleotide sequence of atleast six contiguous nucleotides that is identical to six contiguousnucleotides within the seed region of miR-34a (SEQ ID NO:3), miR-34b(SEQ ID NO:6), and miR-34c (SEQ ID NO:9).

In summary, it appears that the effect of overexpression of miR-449 inp53 wild type cells elicits a phenotype similar to that induced byactivation of the TP53 G1 checkpoint, consistent with the resultsdemonstrated in Example 2 for overexpression of miR-34a, miR-34b, andmiR-34c in A549 cells.

Example 5

This Example demonstrates that introduction of miR-34a causes cell deathin HCT116 Dicer Ex 5 and other cell lines.

Methods:

HCT116 Dicer Ex5 cells were transfected with natural duplexes ofannealed natural miR-34a guide strand (SEQ ID NO:1) and natural miR34apassenger strand (SEQ ID NO:35) or synthetic duplexes of annealednatural miR-34a guide strand (SEQ ID NO:1) and synthetic asymmetricpassenger strand (SEQ ID NO:12) that is complementary to the naturalmature miR-34a, except for a single base mismatch four bases from the 3′end of the sequence (shown in Table 3). Cells were also mocktransfected, or transfected with an siRNA duplex targeting luciferase(SEQ ID NO:27/SEQ ID NO:28). Forty eight hours post transfection, thecells were treated with Nocodazole (100 ng/ml) for 16 hours. Thepercentage of cells in sub-G1 (dead cells) was measured using propidiumiodide staining and flow cytometry.

Results:

TABLE 9 Cell Cycle Arrest in HCT116 Cells (wild type p53) Transfectedwith Natural miR-34a or Synthetic siRNA Constructs microRNA speciesintroduced into % Cells HCT116 dicer−/− cells (wild type p53) in sub-G1miR34a natural (SEQ ID NO: 1/ 27.2% SEQ ID NO: 35) miR-34a mimic (SEQ IDNO: 1/ 43% SEQ ID NO: 12) luciferase control (SEQ ID NO: 27/ 5.7% SEQ IDNO: 28) mock transfection 2.2%

As shown in Table 9, the miR-34a mimic duplex was more effective atinducing cell death than the natural miR-34a duplex, as determined bythe percentage of cells in sub-G1 as measured by flow cytometry oftransfected cells.

While not wishing to be bound by theory, it is believed that thepresence of the mismatch in the asymmetric passenger strand destabilizesthe duplex in that region and thereby facilitates entry into RISC of thestrand mimicking mature miR-34. The duplex miR-34 mimetic sequence withthe asymmetric passenger strand and natural guide strand is processedresulting in formation of the mature wild type miR-34 guide strand.

Summary:

This Example also shows that an siRNA duplex mimetic sequence of miR-34acontaining a natural guide strand annealed to a synthetic passengerstrand that is complementary to the natural mature miR-34a, except for asingle base mismatch four bases from the 3′ end of the sequence,(referred to as “asymmetric passenger strand”) was unexpectedly found tobe more effective at inducing cell death when transfected into cellsthan the natural miR34 duplex.

Example 6

This Example describes the validation of the hepatocyte growth factorreceptor c-MET as a target of miR-34, and the use of synthetic miR-34duplex to inhibit proliferation of the c-MET dependent cell line EBC-1.

Methods:

Activation of c-MET has been implicated in growth, invasion andproliferation in many cancers including non-small cell lung carcinoma(NSCLC), gastric cancer, and a number of lung tumor lines includingEBC-1 depend on c-MET for growth and survival (Lutterbach et al., 2007,Cancer Res/67(5):2081-8).

As shown in TABLE 5, after transfection of synthetic miR-34a, b, or cinto HCT116 Dicer Ex5 cells, a set of genes including the hepatocytegrowth factor receptor (c-MET) was downregulated. This observation wasvalidated by western blotting (data not shown). Consistent with thisobservation, the human c-MET transcript contains two miR-34 target sitesin its ′3 UTR.

To determine the effect of introducing synthetic miR-34 into lung cancercells that are dependent on c-MET for survival, EBC-1 cells (non-smallcell lung cancer) were transfected with a normal synthetic miR-34a RNAduplex (WT mature) comprising a natural guide strand [SEQ ID NO:1] andan asymmetric passenger strand [SEQ ID NO:12] or a seed region doublemutant synthetic miR-34a(2,3) RNA duplex comprising a guide strand [SEQID NO:13] and a passenger strand [SEQ ID NO:14]. 48 hours aftertransfection, the cells were harvested for cell cycle analysis by flowcytometry and Western blot analysis.

Results: EBC-1 cells transfected with normal synthetic miR-34a showed asubstantial increase in sub-G1 population as compared to cellstransfected with seed region mutant synthetic miR-34a or luciferasecontrol. Protein lysates were analyzed by Western blot with antibodiesfor c-MET and cleaved PARP1, an indicator of apoptosis. Cellstransfected with normal synthetic miR-34a showed a decrease in c-METprotein and an increase in cleaved PARP1 in comparison to the cellstransfected with seed region mutant synthetic miR-34a or luciferasecontrol. These results are consistent with the ability of miR-34a tosilence c-MET and induce apoptosis in EBC-1 cells which are dependent onc-MET for survival.

Together, these results demonstrate that a miR-34a therapeutic agentcould be used to inhibit growth and proliferation, and/or promoteapoptosis of c-MET dependent tumors. The use of miR-34a duplexes can bereadily tested in mouse tumor models and xenograft or spontaneous tumorsthat are c-MET dependent.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. An isolated synthetic duplex microRNA mimetic comprising: (i) a guidestrand nucleic acid molecule consisting of a nucleotide sequence of 18to 25 nucleotides, said guide strand nucleotide sequence comprising aseed region nucleotide sequence and a non-seed region nucleotidesequence, said seed region consisting of nucleotide positions 1 to 12and said non-seed region consisting of nucleotide positions 13 to the 3′end of said guide strand, wherein position 1 of said guide strandrepresents the 5′ end of said guide strand, wherein said seed regioncomprises a consecutive nucleotide sequence of at least 6 nucleotidesthat is identical to a seed region sequence of a naturally occurringmicroRNA; and (ii) a passenger strand nucleic acid molecule consistingof a nucleotide sequence of 18 to 25 nucleotides, said passenger strandcomprising a nucleotide sequence that is essentially complementary tothe guide strand, wherein said passenger strand nucleic acid moleculehas one nucleotide sequence difference compared with the true reversecomplement of the seed region of the guide strand, wherein the onenucleotide difference is located within nucleotide position 13 to the 3′end of the passenger strand.
 2. The isolated synthetic duplex microRNAmimetic of claim 1, wherein the guide strand comprises a seed regioncomprising a consecutive nucleotide sequence of at least 6 nucleotidesthat is identical in sequence to a nucleotide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ IDNO:31.
 3. The isolated synthetic duplex microRNA mimetic of claim 1,wherein said guide strand sequence is selected from the group consistingof miR34a (SEQ ID NO:1), miR34b (SEQ ID NO:4), miR34c (SEQ ID NO:7) andmiR449 (SEQ ID NO:29).
 4. The isolated synthetic duplex microRNA mimeticof claim 1, wherein the nucleotide difference in the passenger strand islocated within 6 nucleotides of its 3′ end.
 5. The isolated syntheticduplex microRNA mimetic of claim 1, wherein the synthetic duplex furthercomprises one 3′ overhang, wherein said 3′ overhang comprises from 1 to4 nucleotides.
 6. The isolated synthetic duplex microRNA mimetic ofclaim 5, further comprising a second 3′ overhang wherein said second 3′overhang comprises from 1 to 4 nucleotides.
 7. The isolated syntheticduplex microRNA mimetic of claim 1, wherein said duplex furthercomprises a non-nucleotide moiety.
 8. The isolated synthetic duplexmicroRNA mimetic of claim 1, wherein the guide strand and the passengerstrand are stabilized against nucleolytic degradation.
 9. The isolatedsynthetic duplex microRNA mimetic of claim 1, further comprising atleast one chemically modified nucleotide or non-nucleotide at the 5′ endand/or 3′ end of the guide strand and the 3′ end of the passengerstrand.
 10. The isolated synthetic duplex microRNA mimetic of claim 1,further comprising a phosphorothioate at the first internucleotidelinkage at the 3′ end of the passenger strand and the guide strand. 11.The isolated synthetic duplex microRNA mimetic of claim 1, furthercomprising a phosphorothioate at the first internucleotide linkage atthe 5′ end of the guide stand and the passenger strand, and aphosphorothioate at the first internucleotide linkage at the 3′ end ofthe guide strand and the passenger sequences.
 12. The isolated syntheticduplex microRNA mimetic of claim 1, further comprising a 2′-modifiednucleotide.
 13. The isolated synthetic duplex microRNA mimetic of claim12, wherein the 2′-modified nucleotide comprises a modification selectedfrom the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O-NMA).
 14. A composition comprising at leastone synthetic duplex microRNA mimetic and a delivery agent, thesynthetic duplex microRNA mimetic(s) comprising: (i) a guide strandnucleic acid molecule consisting of a nucleotide sequence of 18 to 25nucleotides, said guide strand nucleotide sequence comprising a seedregion nucleotide sequence and a non-seed region nucleotide sequence,said seed region consisting of nucleotide positions 1 to 12 and saidnon-seed region consisting of nucleotide positions 13 to the 3′ end ofsaid guide strand, wherein position 1 of said guide strand representsthe 5′ end of said guide strand, wherein said seed region furthercomprises a consecutive nucleotide sequence of at least 6 nucleotidesthat is identical in sequence to a seed region of a naturally occurringmicroRNA; and (ii) a passenger strand nucleic acid molecule comprising anucleotide sequence of 18 to 25 nucleotides, said passenger strandcomprising a nucleotide sequence that is essentially complementary tothe guide strand, wherein said passenger strand nucleic acid moleculehas one nucleotide sequence difference compared with the true reversecomplement sequence of the seed region of the guide strand, wherein theone nucleotide difference is located within nucleotide position 13 tothe 3′ end of said passenger strand.
 15. The composition of claim 14,wherein the guide strand comprises a seed region comprising aconsecutive nucleotide sequence of at least 6 nucleotides that isidentical in sequence to a sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:31.
 16. Thecomposition of claim 14, wherein said guide strand sequence is selectedfrom the group consisting of miR34a (SEQ ID NO:1), miR34b (SEQ ID NO:4),miR34c (SEQ ID NO:7) and miR449 (SEQ ID NO:29).
 17. The composition ofclaim 14, wherein the nucleotide difference in the passenger strand islocated within 6 nucleotides of its 3′ end.
 18. The composition of claim14, wherein the delivery agent comprises lipid nanoparticles.